A recombinant human collagen type vi, its production and use

Abstract

The present patent application discloses a recombinant human collagen type VI, a production method and use thereof. The recombinant human collagen type VI is a truncated protein of natural human collagen type VI, and the amino acid sequence is shown as SEQ ID No.: 1. The recombinant human collagen type VI can be efficiently secreted and expressed in a commercial expression system, and the hemostatic and cell adhesion promoting biological activities are higher than those of natural human collagen type VI and other recombinant human collagen type VI.

Description

TECHNICAL FIELD

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

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

[0003] Currently, the collagen used in industrialization is mainly extracted from the skin or bone of animals by acid, alkali or enzyme method, and its main source is animal connective tissue. However, the collagen extracted from animal tissue has the risk of animal source diseases, and large-scale preparation causes great pressure on animal feeding on the supply side.

[0004] With the large-scale application of genetic engineering technology, genetically engineered recombinant expression collagen successfully solves the bottleneck of large-scale preparation of collagen in the form of expression of exogenous protein by mode expression host. Compared with traditional animal collagen extraction, genetically engineered recombinant collagen preparation has the following advantages: 1. The types of produced collagen are various, which can be common bovine, porcine, fish and other source collagen, and also human collagen, which has better safety and immune efficiency in the field of medical devices. 2. Animal source diseases can be effectively avoided. Recombinant collagen usually uses simple prokaryotic or eukaryotic cells as expression host, and the cell pathogen cannot be transmitted due to the huge difference in cell structure with human cells. 3. The production cycle is short, the cost is saved, and the production can be quickly scaled up, which is suitable for industrialized large-scale production. Compared with animal culture which takes 3-5 months, microbial culture cycle only needs 2-3 days and culture is simple. Simple C source and N source can be used for large-scale culture, which is easy for industrial preparation. Therefore, the preparation of recombinant collagen, especially recombinant human collagen, is the research hotspot of collagen production.

[0005] Recombinant human collagen is considered to be the closest collagen to natural synthesis of human body because its amino acid composition is similar or consistent with that of human body. It has good biocompatibility, high safety and the functions of animal collagen. A large number of literatures have reported that commercial products have been successfully developed and applied in the fields of beauty, cosmetics and medical devices.

[0006] Recombinant collagen can be obtained by constructing a truncated protein or a truncated peptide of natural collagen. The uncertainty of the position of the truncated protein in natural collagen and the uncertainty of the length make the number of types of recombinant collagen theoretically several million. However, there is always a lack of effective theoretical guidance on which of these recombinant collagens can have higher secretion yield, and the best results must be screened out through a large number of experimental verifications, which is the main reason for the current few types of recombinant collagen mass production.

[0007] Type VI collagen is widely present in the media and adventitia of arterial smooth muscle, and is co-presented with fibronectin, which may form the final bifurcated shortcut to the basement membrane. At present, the production of type VI collagen mainly has the following problems: 1) natural type VI collagen is difficult to obtain, and the natural type VI collagen is separated from human placenta and purified by 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, and the expression amount 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 not clear, and at present, only the support effect of type VI collagen on cell basement membrane and extracellular matrix is reported, and there is almost no report 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 secreted expression in E. coli) and have clear and prominent biological activity is a technical problem that has always existed in the technical field. SUMMARY

[0009] In view of the technical problems existing in the prior art described above, the purpose of the present application is to provide a new recombinant human type VI collagen, which can be efficiently secreted and expressed in a commercial E. coli expression system, and has good hemostatic and cell adhesion promoting biological activity.

[0010] The inventors have conducted in-depth research to solve the above technical problems, and as a result, it has been found that a truncated protein (221 amino acid residues) from the alpha 2 chain of human natural type VI collagen can be efficiently secreted and expressed in a commercial E. coli expression system (the yield can be as high as about 15 g / L), and has good hemostatic and cell adhesion promoting biological activity, thereby completing the present application.

[0011] That is, the technical scheme of the present application comprises:

[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] GERGDQGGKGDPGRPGRRGPPGEIGAKGSKGYQGNSGAPGSPGVKGAK

[0015] GGPGPRGPKGEPGRRGDPGTKGSPGSDGPKGEKGDPGPEGPRGLAGEVG

[0016] NKGAKGDRGLPGPRGPQGALGEPGKQGSRGDPGDAGPRGDSGQPGPKG

[0017] DPGRPGFSYPGPRGAPGEKGEPGPRGPEGGRGDFGLKGEPGRKGEKGEPA

[0018] DPGPPGEPGPRGPRGVPGPEGEPGPP

[0019] 2. A nucleic acid encoding the recombinant human collagen type VI according to claim 1.

[0020] 3. An expression vector comprising the nucleic acid according to claim 2.

[0021] 4. A host cell into which the expression vector according to claim 3 is introduced. The host cell can be a prokaryotic cell or a eukaryotic cell, including bacterial hosts such as Escherichia coli, Bacillus subtilis, Bacillus licheniformis, and the like, eukaryotic hosts such as Pichia pastoris, Saccharomyces cerevisiae, animal cells, plant cells, and the like, preferably Escherichia coli and Pichia pastoris, and more preferably Escherichia coli.

[0022] 5. A method for producing the recombinant human collagen type VI according to claim 1, comprising the step of culturing the host cell according to claim 4 so that it expresses the recombinant human collagen type VI according to claim 1, and collecting the culture containing the recombinant human collagen type VI. The expression can be one of constitutive expression, inducible expression, or a combination of both. The inducer for inducible expression can be IPTG, β-galactoside, methanol, ethanol, and the like.

[0023] 6. The production method according to claim 5, further comprising the step of subjecting the culture to separation and purification, thereby obtaining the purified recombinant human collagen type VI.

[0024] 7. The production method according to claim 6, wherein the separation and purification employs one or more of a combination of salting-out, chromatography, affinity chromatography, acid-base precipitation, membrane separation, preferably a combination of chromatography and membrane separation, or a combination of ion exchange chromatography and membrane separation.

[0025] 8. Use of the recombinant human collagen type VI according to claim 1 in the preparation of skin care products, health care products, tissue engineering materials or medical devices.

[0026] 9. The use according to claim 8, wherein the tissue engineering material is selected from the group consisting of subcutaneous fillers, artificial bone, artificial skin, oral absorbable biological membrane, bone implant, vascular stent, hemostatic agent, procoagulant agent, cellular interstitial stent and collagen sponge.

[0027] The present application has the beneficial effect of providing a recombinant collagen type VI which can be efficiently secreted and expressed in a commercial E. coli expression system, and has good hemostatic and cell adhesion promoting biological activities. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 It is an electrophoresis map showing the induction fermentation results of the recombinant collagen protein (2 ul loading amount).

[0029] Figure 2 It is an electrophoresis map showing the results after purification of the recombinant collagen protein (2 ul loading amount). DETAILED DESCRIPTION

[0030] Example 1 Preparation of recombinant collagen protein VI-1 in E. coli expression system

[0031] I. Experimental methods

[0032] 1. Preparation of shuttle plasmid and E. coli expression strain

[0033] According to the amino acid sequence shown in SEQ ID No. 1, the codon optimization of the E. coli expression system was carried out to obtain the target gene sequence of the recombinant collagen protein VI-1. The obtained target gene sequence was entrusted to GenScript Biotech Co., Ltd. for gene synthesis. The synthesized gene was connected to the pet-28a plasmid to obtain the pET28a-VI-1 plasmid.

[0034] The pET28a-VI-α2 plasmid was transformed into E. coli competent cells, and kanamycin resistance was used as a screening marker to screen the transformants, thereby obtaining the E. coli expression strain.

[0035] 2. Induced expression of target protein

[0036] (1) A single colony of the constructed E. coli expression strain was picked and added to 5 ml of LB liquid medium (0.5% yeast extract, 1% peptone and 1% sodium chloride), and incubated at 37°C, 200 rpm for 18 h for activation;

[0037] (2) 500ml flask (200ml LB medium) was inoculated with 1% of the inoculum, and incubated at 37°C and 200rpm for 12h as the seed for the upper tank;

[0038] (3) High-density fermentation medium was prepared, and the medium composition was as follows:

[0039] Yeast powder 20-60g / L, glycerol 20-40g / L, yeast extract 5-10g / L, potassium phosphate dibasic trihydrate 3.4-6.8g / L, potassium phosphate monobasic 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.

[0040] 3L of the medium was added to a 5L fermenter, sterilized at 121°C for 20min, and then cooled to 37°C. The pH was adjusted to 6.8, and the seed prepared in (2) was added to the fermenter by flame inoculation for fermentation culture;

[0041] (4) The culture was incubated to OD 600 30 or so, and when the dissolved oxygen rebounded to 100%, 50% glycerol and 20% yeast powder were added for induction, and 1mMol / L IPTG was added.

[0042] (5) During the induction process, the dissolved oxygen was controlled to be not less than 30%, and the pH was about 6.8. After 12h of induction, the fermentation was completed. The culture solution was centrifuged at 8000rpm for 20min, and the supernatant was removed. The bacteria were retained and diluted to a certain multiple. The protein diluent was obtained by ultrasonic crushing method. BCA method and SDS-PAGE method were used to detect the protein yield and purity.

[0043] 3. Collagen purification

[0044] The collected protein diluent was passed through a 15KD ultrafiltration membrane, and then passed through a hollow fiber column and freeze-dried to obtain recombinant collagen VI-1.

[0045] II. Experimental results

[0046] The fermentation tank yield of the recombinant collagen VI-1 prepared by the above method was as high as 30g / L by BCA detection. The electrophoresis detection result by SDS-PAGE method showed that the target protein accounted for more than 50%. The target protein yield in the fermentation broth could reach 15g / L. The protein purity after passing through the hollow fiber column could reach more than 80%. The SDS-PAGE electropherogram of the protein before purification is shown in Figure 1, and the SDS-PAGE electrophoretogram of the purified protein is shown in Figure 2 .

[0047] Example 2 Preparation of recombinant collagen VI in an E. coli expression system

[0048] Using the same design idea as the recombinant collagen VI-1, a protein sequence with similar molecular weight was cut from the natural collagen VI α2 chain, and was expressed in the same way. The sequence design and expression results are shown in the following table (Table 1), and the SDS-PAGE electrophoretogram of the protein before purification is shown in Figure 1 , and the SDS-PAGE electrophoretogram of the purified protein is shown in Figure 2 .

[0049] Table 1 Different amino acid sequences and expression results

[0050]

[0051] 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.

[0052] As can be seen from Table 1, the yield of the recombinant collagen VI-1 is much higher than that of the similarly designed recombinant collagen VI-2 and VI-3, and the purity of the target protein is also higher. Therefore, the recombinant collagen VI-1 provided by the present application is easier to prepare on a large scale and has more potential for mass production.

[0053] Example 3: Application of recombinant collagen VI in hemostasis

[0054] Specific implementation method:

[0055] Establishment of liver hemorrhage animal model and evaluation of hemostatic effect 50 SD rats were randomly divided into 3 groups according to the body weight of the animals using a random number table method, and the sample size of the animals was determined by an unpaired experimental method. Among them, 30 experimental group rats used recombinant VI-1 collagen protein, VI-2 collagen protein and VI-3 collagen protein as filling materials for liver damage, 10 control group rats used hemostatic sponge as filling materials for liver damage, and 10 blank group rats were not filled after partial liver resection.

[0056] Construction of liver hemorrhage rat model: The animals were fasted for at least 2 hours before surgery, and 1% sodium pentobarbital was injected intraperitoneally. After satisfactory anesthesia, the abdominal surgical area was prepared, iodophor was used for disinfection, and the abdominal cavity was opened layer by layer to expose the liver. A sterile cotton swab was used to wipe the abdominal cavity, abdominal wall and peritoneal fluid around the liver clean, and sterile gauze was weighed and placed under the liver. A pair of ophthalmic surgical scissors was used to cut 1cm 2The incision was made to successfully cut the liver and visible bleeding was judged to be successful modeling, and the time was recorded. Different groups were filled with corresponding materials to fill the wound, and the blank group was not filled with materials. The hemostatic time was recorded, and the gauze was removed and weighed after hemostasis. The abdominal cavity was sutured layer by layer, iodophor was sterilized, penicillin sodium was intramuscularly injected, and the mental state, activity and skin healing of the rats were observed for 3 consecutive days. The abnormal reactions of the rats, the presence or absence of exudate in the abdominal cavity and the infection were recorded in detail.

[0057] Hemostatic effect evaluation: ①Hemostatic time: the time was recorded when the liver was cut, and the time was recorded when the bleeding stopped. ②Bleeding volume: the gauze was weighed and placed under the liver, and the gauze under the liver was weighed again after the bleeding stopped. The difference between the two gauze weights was the bleeding volume.

[0058] The results are as follows:

[0059] Table 2: Hemostatic time and bleeding weight of rats in each group

[0060] 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

[0061] As shown in Table 2, the recombinant collagen VI-1 can significantly shorten the bleeding time and reduce the bleeding volume compared with other recombinant collagen VI, and has good hemostatic effect.

[0062] Example 4: Application of recombinant collagen VI in preparing cell adhesion promoter

[0063] Specific implementation method:

[0064] Normal HSF (human skin fibroblast) cells were cultured, and the freeze-dried product of the purified recombinant collagen VI was dissolved in ultrapure water, and then diluted with PBS solution to 0.25 mg / L, 0.5 mg / L and 1 mg / L. The HSF (human skin fibroblast) cells were treated with the above solutions for cell adhesion promotion experiment. The specific steps are as follows: the human HSF cells were inoculated in a sterile 96-well plate, 200 μL of culture medium was added to each well, and the blank control group was only added with PBS. After being placed at 37℃ for 2 h, each group was washed with PBS for 3 times to wash away the unadhered cells. 100 μL of culture medium and 50 μL of MTT solution were added to each well, shaken, and then incubated in a culture box for 4 h. After the mixed solution was aspirated, 150 μL of DMSO was added to dissolve the purple crystal formazan, shaken for 10 min, and then placed in an enzyme marker instrument for detection. The detection wavelength was 570 nm, and the absorbance value of each well was measured.

[0065] The data of the recombinant collagen VI and natural collagen VI (purchased from coriong Matrigel company in USA) promoting HSF (human skin fibroblast) cell adhesion are shown in Table 1, wherein the cell adhesion promoting rate = (OD of experimental group - OD of blank group) x 100% / blank group.

[0066] Table 3 cell adhesion promoting rate values of different concentrations of recombinant collagen on skin fibroblast cell adhesion promotion

[0067]

[0068] The results show that the recombinant collagen VI-1 can significantly promote HSF cell adhesion compared to other recombinant collagen VI, and the adhesion promoting effect is also significantly higher than that of natural human collagen VI, has good cell compatibility, and shows good cell adhesion promoting effect.

[0069] Sequence information

[0070] SEQ ID No. 1

[0071] GERGDQGGKGDPGRPGRRGPPGEIGAKGSKGYQGNSGAPGSPGVKGAKGGPGPRGPKGEPGRRGDPGT

[0072] KGSPGSDGPKGEKGDPGPEGPRGLAGEVGNKGAKGDRGLPGPRGPQGALGEPGKQGSRGDPGDAGPRG

[0073] DSGQPGPKGDPGRPGFSYPGPRGAPGEKGEPGPRGPEGGRGDFGLKGEPGRKGEKGEPADPGPPGEPGPR

[0074] GPRGVPGPEGEPGPP

[0075] SEQ ID No. 2

[0076] DIASTPHELYRNDYATMLPDSTEIDQDTINRIIKVMKHEAYGECYKVSCLEIPGPSGPKGYRGQKGAKGNM

[0077] GEPGEPGQKGRQGDPGIEGPIGFPGPKGVPGFKGEKGEFGADGRKGAPGLAGKNGTDGQKGKLGRIGPP

[0078] GCKGDPGNRGPDGYPGEAGSPGERGDQGGKGDPGRPGRRGPPGEIGAKGSKGYQGNSGAPGSPGVKGA

[0079] KGGPGPRGPKGEPGRRG

[0080] SEQ ID No. 3

[0081] PGDAGPRGDSGQPGPKGDPGRPGFSYPGPRGAPGEKGEPGPRGPEGGRGDFGLKGEPGRKGEKGEPADP

[0082] GPPGEPGPRGPRGVPGPEGEPGPPGDPGLTECDVMTYVRETCGCCDCEKRCGALDVVFVIDSSESIGYTNF

[0083] TLEKNFVINVVNRLGAIAKDPKSETGTRVGVVQYSHEGTFEAIQLDDERIDSLSSFKEAVKNLEWIAGGT

[0084] WTPSALKFAYDR

Claims

1. A recombinant human collagen type VI having an amino acid sequence as set forth in SEQ ID No.

1. 2.A nucleic acid encoding the recombinant human collagen type VI of claim 1. 3.An expression vector comprising the nucleic acid of claim 2. 4.A host cell into which the expression vector of claim 3 is introduced. 5.The host cell of claim 4, which is Escherichia coli or Pichia pastoris. 6.A method of producing the recombinant human collagen type VI of claim 1, comprising the step of culturing the host cell of claim 4 so that it expresses the recombinant human collagen type VI of claim 1, and collecting the culture containing the recombinant human collagen type VI. 7.The method of production of claim 6, further comprising the step of subjecting the culture to separation and purification, thereby obtaining the purified recombinant human collagen type VI.

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