A stable recombinant collagen type i and its use

Abstract

The application provides a stable recombinant collagen type I and application thereof, the recominat collagen type I is designed based on the amino acid sequence of human collagen type I, and includes 19 basic units in series, the amino acid sequence of the basic unit is shown as SEQ ID NO.1, and the amino acid sequence of the recominat collagen type I is shown as SEQ ID NO.6.The application also establishes a high-efficiency recombinant expression system and a label-free protein purification process, avoids the potential influence of foreign label residues on the natural conformation and functional integrity of the protein, makes the recominat collagen type I closer to the collagen of human body in structure and biological activity, has high purity, excellent water solubility and stability, and improves the reliability of clinical application.The application lays a solid foundation for realizing stable and low-cost industrial production of collagen type I, and helps to meet the wide market demand for high-quality collagen.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to a stable recombinant type I collagen and its applications. Background Technology

[0002] Type I collagen is a key functional component of the human extracellular matrix, possessing irreplaceable application value in the medical field due to its excellent biocompatibility, cell adhesion activity, and tissue repair capabilities. However, existing technologies for obtaining and preparing type I collagen face significant bottlenecks, severely restricting its high-quality, large-scale industrial application.

[0003] Animal tissue extraction is the main method for obtaining type I collagen, but this process is complex, and the resulting product is animal-derived, which may trigger immune rejection and carries the risk of viral contamination. Furthermore, batch-to-batch quality variations are significant, controllability is poor, and large-scale production is limited, making it difficult to meet the high standards of material consistency and safety required by modern medicine.

[0004] The rise of genetic engineering technology has provided a new direction for the production of type I collagen. However, type I collagen prepared by recombinant expression technology currently faces the challenge of insufficient stability, and is prone to denaturation, degradation, or loss of function during storage and application. Therefore, there is an urgent need in this field to develop a new generation of preparation process that can safely, efficiently, with high purity, and stably scale up the production of type I collagen by integrating human type I collagen sequences, in order to fill the urgent demand gap for high-quality collagen raw materials in the biotechnology, medical, and other industries. Summary of the Invention

[0005] To address the aforementioned problems in the prior art, this invention provides a stable recombinant type I collagen and its applications.

[0006] In a first aspect, the present invention provides a recombinant type I collagen, the recombinant type I collagen comprising the amino acid sequence shown in SEQ ID NO. 6.

[0007] According to the present invention, a recombinant type I collagen is preferably provided, wherein the recombinant type I collagen does not include a label.

[0008] More preferably, the label is selected from at least one of the His label, GST label, MBP label, Strep label and FLAG label.

[0009] More preferably, the label is a His label.

[0010] More preferably, the amino acid sequence of the recombinant type I collagen has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO. 6 and has the same function.

[0011] In some specific embodiments, the amino acid sequence of the recombinant type I collagen is shown in SEQ ID NO.6.

[0012] In a second aspect, the present invention provides a nucleic acid molecule encoding recombinant type I collagen, said recombinant type I collagen comprising the amino acid sequence shown in SEQ ID NO. 6.

[0013] According to a nucleic acid molecule provided by the present invention, preferably, the recombinant type I collagen does not include a tag.

[0014] More preferably, the label is selected from at least one of the His label, GST label, MBP label, Strep label and FLAG label.

[0015] More preferably, the label is a His label.

[0016] More preferably, the amino acid sequence of the recombinant type I collagen has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO. 6 and has the same function.

[0017] In some specific embodiments, the amino acid sequence of the recombinant type I collagen is shown in SEQ ID NO.6.

[0018] According to the present invention, a nucleic acid molecule preferably comprises a nucleotide sequence as shown in SEQ ID NO.7.

[0019] More preferably, the nucleotide sequence of the nucleic acid molecule has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the nucleotide sequence shown in SEQ ID NO.7 and encodes a protein with the same function.

[0020] In some specific embodiments, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.7.

[0021] Thirdly, the present invention provides a biological material comprising the aforementioned nucleic acid molecule; the biological material is recombinant DNA, expression cassette, transposon, vector, cell, or microorganism.

[0022] According to a biomaterial provided by the present invention, preferably, the vector uses a yeast expression vector as a backbone.

[0023] More preferably, the carrier uses a pPICZαA carrier or a pPIC9k carrier as its backbone.

[0024] More preferably, the carrier uses the pPICZαA carrier as its backbone.

[0025] More preferably, the nucleic acid molecule is located between the EcoR I site and the Not I site of the backbone.

[0026] According to the present invention, the microorganisms preferably include yeast.

[0027] More preferably, the yeast is Pichia pastoris.

[0028] More preferably, the Pichia pastoris is Pichia pastoris strain X33, Pichia pastoris strain GS115, Pichia pastoris strain KM71, or Pichia pastoris strain SMD1168.

[0029] More preferably, if the vector uses pPICZαA vector as the backbone, then the Pichia pastoris is Pichia pastoris strain X33.

[0030] More preferably, if the vector uses the pPIC9k vector as a backbone, then the Pichia pastoris is Pichia pastoris strain GS115, Pichia pastoris strain KM71, or Pichia pastoris strain SMD1168.

[0031] In a further preferred embodiment, the vector uses the pPICZαA vector as its backbone, the Pichia pastoris is the Pichia pastoris X33 strain, and the nucleic acid molecule is located between the EcoR I site and the Not I site of the pPICZαA vector.

[0032] Fourthly, the present invention provides a conjugate comprising the recombinant type I collagen and a chemical moiety conjugated thereto.

[0033] A composition comprising the recombinant type I collagen described above should also be within the scope of protection of this invention.

[0034] Fifthly, the present invention provides the application of the recombinant type I collagen, the nucleic acid molecule, the biomaterial, or the conjugate in the preparation of drugs, medical biomaterials, or medical devices.

[0035] In a sixth aspect, the present invention provides a product containing the recombinant type I collagen, or containing the nucleic acid molecule, or containing the biomaterial, or containing the conjugate, or containing the composition; the product is a pharmaceutical, medical biomaterial, or medical device.

[0036] In a seventh aspect, the present invention provides a method for preparing recombinant type I collagen, comprising: culturing microorganisms in the biological material while allowing the recombinant type I collagen to be expressed, and recovering the recombinant type I collagen from the culture product.

[0037] According to the present invention, a method for preparing recombinant type I collagen is provided, wherein preferably, the carrier uses a yeast expression vector as the backbone.

[0038] More preferably, the carrier uses a pPICZαA carrier or a pPIC9k carrier as its backbone.

[0039] More preferably, the carrier uses the pPICZαA carrier as its backbone.

[0040] More preferably, the nucleic acid molecule is located between the EcoR I site and the Not I site of the backbone.

[0041] According to the method for preparing recombinant type I collagen provided by the present invention, preferably, the microorganisms include yeast.

[0042] More preferably, the yeast is Pichia pastoris.

[0043] More preferably, the Pichia pastoris is Pichia pastoris strain X33, Pichia pastoris strain GS115, Pichia pastoris strain KM71, or Pichia pastoris strain SMD1168.

[0044] More preferably, if the vector uses the pPICZαA vector as the backbone, then the Pichia pastoris is the Pichia pastoris X33 strain.

[0045] More preferably, if the vector uses the pPIC9k vector as a backbone, then the Pichia pastoris is Pichia pastoris strain GS115, Pichia pastoris strain KM71, or Pichia pastoris strain SMD1168.

[0046] In a further preferred embodiment, the vector uses the pPICZαA vector as its backbone, the Pichia pastoris is the Pichia pastoris X33 strain, and the nucleic acid molecule is located between the EcoR I site and the Not I site of the pPICZαA vector.

[0047] According to the method for preparing recombinant type I collagen provided by the present invention, preferably, the culture temperature is 25℃~35℃.

[0048] More preferably, the culture temperature is 30°C.

[0049] Sorbitol is a highly efficient auxiliary carbon source and metabolic regulator in the induction medium of Pichia pastoris. More preferably, the medium used for the induction culture of said Pichia pastoris includes BMMY medium containing sorbitol. Sorbitol exerts the following effects: 1. Alleviating methanol toxicity and enhancing the cell viability and stability of Pichia pastoris; 2. Optimizing energy metabolism and reducing process load; 3. Promoting proper protein folding and secretion, increasing activity and yield; 4. Reducing oxidative modification and improving product quality.

[0050] More preferably, the working concentration of sorbitol is 0.5% w / v to 3% w / v.

[0051] More preferably, the working concentration of sorbitol is 2% w / v.

[0052] According to a method for preparing recombinant type I collagen provided by the present invention, preferably, the method further includes: purifying the recovered recombinant type I collagen, wherein the purification method is selected from any one or more of affinity chromatography, ion exchange, hydrophobic chromatography, chromatographic chromatography and salting out.

[0053] More preferably, the purification method is ion exchange.

[0054] More preferably, the ion exchange is performed using a strong cation exchange column.

[0055] More preferably, the packing material of the strong cation exchange column is sulfopropyl packing material.

[0056] More preferably, the recovered recombinant type I collagen is mixed with 0.4% v / v to 0.6% v / v acetic acid at a volume ratio of 1:(1 to 4), and then purified using a strong cation exchange chromatography column.

[0057] More preferably, the recovered recombinant type I collagen is mixed with 0.5% v / v acetic acid at a volume ratio of 1:(1-4), and then purified using a strong cation exchange chromatography column.

[0058] More preferably, the recovered recombinant type I collagen is mixed with 0.4% v / v to 0.6% v / v acetic acid at a volume ratio of 1:1, and then purified using a strong cation exchange chromatography column.

[0059] More preferably, the recovered recombinant type I collagen is mixed with 0.5% v / v acetic acid at a volume ratio of 1:1, and then purified using a strong cation exchange chromatography column.

[0060] More preferably, the recombinant type I collagen is eluted from the strong cation exchange column using an eluent containing 40 mM to 60 mM NaCl.

[0061] More preferably, the recombinant type I collagen is eluted from the strong cation exchange column using an eluent containing 50 mM NaCl.

[0062] More preferably, the recombinant type I collagen is eluted from the strong cation exchange column using an eluent containing 10 mM to 30 mM PB.

[0063] More preferably, the recombinant type I collagen is eluted from the strong cation exchange column using an eluent containing 20 mM PB.

[0064] More preferably, the pH of the eluent is 6.0 to 7.0.

[0065] More preferably, the pH of the eluent is 6.5.

[0066] More preferably, the UV absorbance of the eluted product is monitored in real time during the elution process with the elution buffer, and the eluted product corresponding to the first protein peak at A280 nm is the purified product of the recombinant type I collagen.

[0067] More preferably, the preparation method further includes: dialysis of the purified product of the recovered recombinant type I collagen.

[0068] More preferably, the dialysis is performed using a dialysis bag with a molecular weight cutoff of 3.5 kDa.

[0069] More preferably, the buffer solution used for dialysis is 18mM to 22mM PB containing 40mM to 60mM NaCl, with a pH of 6.0 to 7.0.

[0070] More preferably, the buffer solution used for dialysis is 20mM PB containing 50mM NaCl, with a pH of 6.5.

[0071] The present invention has the following beneficial effects:

[0072] The recombinant type I collagen prepared by this invention is designed based on the amino acid sequence of human type I collagen, eliminating the risks of immune rejection and allergic reactions that may be caused by animal-derived collagen, thus providing a higher level of safety for its clinical application. This invention also establishes an efficient recombinant expression system and a tag-free protein purification process, avoiding the potential impact of exogenous tag residues on the protein's native conformation and functional integrity. This makes the recombinant type I collagen structurally and biologically closer to human collagen, with high purity and excellent water solubility and stability, improving the reliability of clinical applications. This lays a solid foundation for the stable, low-cost, large-scale industrial production of type I collagen, helping to meet the widespread market demand for high-quality collagen. Attached Figure Description

[0073] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0074] Figure 1 This is the hydrophilicity prediction result of the basic unit 1 (SEQ ID NO.1) provided in Embodiment 1 of the present invention.

[0075] Figure 2 This is the hydrophilicity prediction result of the basic unit 2 (SEQ ID NO.2) provided in Embodiment 1 of the present invention.

[0076] Figure 3 This is the hydrophilicity prediction result of the basic unit 3 (SEQ ID NO.3) provided in Embodiment 1 of the present invention.

[0077] Figure 4 This is the hydrophilicity prediction result of the basic unit 4 (SEQ ID NO.4) provided in Embodiment 1 of the present invention.

[0078] Figure 5 This is the hydrophilicity prediction result of the basic unit 5 (SEQ ID NO.5) provided in Embodiment 1 of the present invention.

[0079] Figure 6 This is a spectrum of the recombinant plasmid pPICZαA-CollagenⅠ provided in Example 2 of the present invention.

[0080] Figure 7 This is a spectrum of the recombinant plasmid pPIC9k-CollagenⅠ provided in Example 2 of the present invention.

[0081] Figure 8This is the result of double enzyme digestion identification of the recombinant plasmid pPICZαA-CollagenⅠ provided in Example 2 of the present invention.

[0082] Figure 9 This is the result of double enzyme digestion identification of the recombinant plasmid pPIC9k-CollagenⅠ provided in Example 2 of this invention.

[0083] Figure 10 The results of SDS-PAGE electrophoresis of the recombinant Pichia pastoris strain X33 provided in Example 3 of this invention are shown below; M is the protein molecular weight standard, and the bands from largest to smallest are 140 kDa, 115 kDa, 80 kDa, 65 kDa, 50 kDa, 40 kDa, 30 kDa, 25 kDa, 15 kDa and 10 kDa; lanes 1 to 10 correspond to 10 single colonies pPICZαA-CollagenⅠ / X33-1 to pPICZαA-CollagenⅠ / X33-10 respectively.

[0084] Figure 11 This is an example of SDS-PAGE electrophoresis used in Example 4 of the present invention to detect the expression of recombinant type I collagen at different induction times; M is the protein molecular weight standard, and the bands from largest to smallest are 140 kDa, 115 kDa, 80 kDa, 65 kDa, 50 kDa, 40 kDa, 30 kDa, 25 kDa, 15 kDa and 10 kDa; lanes 1 to 3 correspond to induction times of 24 h, 48 h and 72 h, respectively.

[0085] Figure 12 This is an example of SDS-PAGE electrophoresis used to detect the purification status of recombinant type I collagen in Example 4 of the present invention; lanes 1 to 7 are, in order, fermentation supernatant (diluted 4 times with 0.5% acetic acid), FT, EpH4.0, EpH5.0, EpH6.0, E50-1 and E50-2.

[0086] Figure 13 This is the stability evaluation result of the recombinant type I collagen provided in the test examples of this invention. Detailed Implementation

[0087] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0088] Unless otherwise specified, the experimental methods involved in the following embodiments are conventional methods in the art. For example, you can refer to the experimental manual in the art or follow the conditions recommended in the manufacturer's instructions.

[0089] Unless otherwise specified, all experimental materials and reagents used in the following examples are commercially available.

[0090] The following examples used Pichia pastoris strain X33, Pichia pastoris strain GS115, Pichia pastoris strain KM71, and Pichia pastoris strain SMD1168, all of which were preserved by Jinpro Protein Engineering Technology (Beijing) Co., Ltd. Restriction endonucleases EcoRI and NotI were purchased from NEB (Beijing) Co., Ltd. Tryptone, yeast extract, bleomycin, and BCA protein quantification kits were purchased from Thermo Fisher Scientific. Protein electrophoresis buffer, staining solution, and destaining solution were purchased from Genscript Biotech Co., Ltd. All other reagents were domestically produced analytical grade.

[0091] The formulations of the culture media used in the following examples are as follows: (1) YPDS solid medium (100ml): Dissolve 1g yeast extract and 2g peptone in 90ml water; autoclave for 20min; add 10mL 20% glucose, 18.22g sorbitol and 2g agar powder, and dissolve thoroughly; (2) BMGY liquid medium (1L): Dissolve 10g yeast powder and 20g peptone in 700ml water, autoclave for 20min; cool to room temperature, add 100ml 1M potassium phosphate buffer pH6.0 (room temperature), 100ml 13.4% yeast nitrogen base (YNB), 100ml 10% glycerol (GyY) and 2ml 0.02% Biotin, mix thoroughly; (3) BMMY liquid medium (1L): Dissolve 10g yeast powder and 20g peptone in 700ml water, sterilize for 20min; cool to room temperature, add 100ml 1M potassium phosphate buffer pH6.0 (room temperature), 100ml 13.4% YNB, 100ml 5% methanol (Methanol, M) and 2ml 0.02% Biotin, mix thoroughly; (4) YPD solid medium (100ml): Dissolve 1g yeast extract and 2g peptone in 90ml water; autoclave for 20min; add 10mL 20% glucose and 2g agar powder, dissolve thoroughly; (5) MD solid medium (100ml): Dissolve 2g agar powder in 80ml water, autoclave for 20min; cool to room temperature, add 10ml 13.4% YNB, 200μl 0.02% Biotin, 10ml 20% glucose.

[0092] Example 1: Determination of the amino acid sequence of recombinant type I collagen

[0093] 1. Screening of basic unit amino acid sequences

[0094] Based on the amino acid sequence characteristics of human type I collagen, this invention screened out five candidate basic units, whose amino acid sequences are as follows:

[0095] Basic Unit 1: GASGPMGPRGPPGPPGKNGDDGEAGKPGRPGERGPPGPQGARGLP (SEQ ID NO.1);

[0096] Basic Unit 2: QLSYGYDEKSTGGISVPGPMGPSGPRGLPGPPGAPGPQGFQGPPG (SEQ ID NO.2);

[0097] Basic Unit 3: GAPGSKGDTGAKGEPGPVGVQGPPGPAGEEGKRGARGEPGPTGLP (SEQ ID NO.3);

[0098] Basic Unit 4: GDAGPVGPPGPPGPPGPPGPPSAGFDFSFLPQPPQEKAHDGGRYY (SEQ ID NO.4);

[0099] Basic unit 5: GVEGPKGDTGPRGPRGPAGPPGRDGIPGQPGLPGPPGPPGPPGPP (SEQ ID NO.5).

[0100] 2. Evaluation of the hydrophilicity / hydrophobicity of basic units

[0101] (1) Evaluation method

[0102] The hydrophilicity of five candidate basic units (amino acid sequences shown in SEQ ID NO. 1–5) of recombinant type I collagen was predicted using the website https: / / web.expasy.org / protscale / .

[0103] (2) Evaluation results

[0104] like Figures 1-5 As shown, compared to other candidate basic units, basic unit 1 (SEQ ID NO.1) has no hydrophobic regions, indicating that it has the strongest hydrophilicity and extremely high solubility.

[0105] 3. Stability evaluation of basic units

[0106] (1) Evaluation method

[0107] The stability of five candidate basic units (amino acid sequences shown in SEQ ID NO. 1–5) of recombinant type I collagen was predicted using the website https: / / web.expasy.org / protparam / .

[0108] (2) Evaluation results

[0109] The results showed that the instability indices of basic units 1 to 5 were 10.53, 33.07, 25.11, 66.52 and 27.25, respectively, indicating that basic unit 1 (SEQ ID NO.1) had the lowest instability index compared to other candidate basic units, indicating that it had the strongest stability.

[0110] Based on the above evaluation results, the basic unit 1 with the amino acid sequence shown in SEQ ID NO.1 was selected to construct a recombinant plasmid expressing recombinant type I collagen.

[0111] Example 2: Construction and identification of recombinant plasmids expressing recombinant type I collagen

[0112] 1. Construction of recombinant plasmids

[0113] This invention designs a repeating tandem sequence based on the amino acid sequence characteristics of human type I collagen. The basic unit 1 (SEQ ID NO.1) from Example 1 is repeated tandemly 19 times, and then a glycine-proline-proline tripeptide (Gly-Pro-Pro, GPP) is attached to the N-terminus and C-terminus respectively, resulting in the amino acid sequence shown in SEQ ID NO.6, which is the complete sequence of recombinant type I collagen. The nucleotide sequence of its corresponding encoding gene is shown in SEQ ID NO.7.

[0114] The coding gene sequence (SEQ ID NO.7) was synthesized by General Biotechnology (Anhui) Co., Ltd. to obtain the CollagenⅠ gene, which was then ligated into the Pichia pastoris expression vectors pPICZαA and pPIC9k, respectively, to obtain the corresponding results as shown below. Figure 6 and Figure 7 The recombinant plasmids shown are named pPICZαA-CollagenⅠ and pPIC9k-CollagenⅠ, respectively.

[0115] 2. Identification of recombinant plasmids

[0116] Two µl of pPICZαA-CollagenⅠ and two µl of pPIC9k-CollagenⅠ were identified by double digestion with EcoRI and Not I, and the digestion products were collected for 1% agarose gel electrophoresis.

[0117] like Figure 8 As shown, after double digestion of pPICZαA-CollagenⅠ, two amplification bands of 3551bp and 2586bp were observed.

[0118] like Figure 9 As shown, after double digestion of pPIC9k-CollagenⅠ, two amplified bands of 9270bp and 2586bp were observed.

[0119] This indicates that the CollagenⅠ gene has been successfully ligated into the pPICZαA and pPIC9k vectors, respectively, and the two recombinant plasmids have been successfully constructed.

[0120] Example 3 Construction and identification of recombinant strains expressing recombinant type I collagen

[0121] 1. Transformation of recombinant plasmid pPICZαA-CollagenⅠ and screening of high-copy transformants

[0122] pPICZαA-CollagenⅠ was transformed into competent cells of strain X33, and the cells were spread on the surface of YPDS solid medium containing 200 μg / ml bleomycin. After standing at 30°C for 20 min, the cells were incubated upside down for 2–3 days. Single colonies grown on the plates were transferred to YPD solid medium containing 200 μg / ml bleomycin for high-copy transformant screening.

[0123] The method for screening high-copy transformants of pPICZαA-CollagenⅠ is as follows:

[0124] Colonies were picked from YPDS solid medium plates containing 200 μg / ml bleomycin, transferred to YPD solid medium plates containing 200 μg / ml bleomycin, and numbered. The plates were then incubated upside down at 30°C for 16–18 h. Subsequently, 10 single colonies were picked from each YPD solid medium plate and inoculated into 10 ml of BMGY liquid medium. After incubation at 30°C and 280 rpm for 24 h, samples were taken and examined under a microscope to confirm the absence of contamination.

[0125] The 10 single colonies of the obtained recombinant Pichia pastoris strain X33 were named pPICZαA-CollagenⅠ / X33-1 to pPICZαA-CollagenⅠ / X33-10.

[0126] 2. Transformation of recombinant plasmid pPIC9k-CollagenⅠ and screening of high-copy transformants

[0127] pPIC9k-CollagenⅠ was transformed into competent cells of strains GS115, KM71 and SMD1168, respectively, and then plated onto the surface of MD solid medium plates. After standing at 30°C for 20 min, the plates were inverted and cultured for 2-3 days. Single colonies grown on the plates were transferred to another MD solid medium plate for high copy transformation screening.

[0128] The method for screening high-copy transformants of pPIC9k-CollagenⅠ is as follows:

[0129] Colonies were picked from MD solid medium plates, transferred to another MD solid medium plate and numbered, and incubated upside down at 30°C for 16–18 h. Then, for each strain, 10 single colonies were picked from each MD solid medium plate and inoculated into 10 ml of BMGY liquid medium. After incubation at 30°C and 280 rpm for 24 h, samples were taken and examined under a microscope to confirm the absence of contamination.

[0130] The 10 single colonies of the obtained recombinant Pichia pastoris strain GS115 were named pPIC9k-CollagenⅠ / GS115-1 to pPIC9k-CollagenⅠ / GS115-10, the 10 single colonies of the obtained recombinant Pichia pastoris strain KM71 were named pPIC9k-CollagenⅠ / KM71-1 to pPIC9k-CollagenⅠ / KM71-10, and the 10 single colonies of the obtained recombinant Pichia pastoris strain SMD1168 were named pPIC9k-CollagenⅠ / SMD1168-1 to pPIC9k-CollagenⅠ / SMD1168-10.

[0131] 3. Colony identification

[0132] The bacterial cultures of each single colony of the four recombinant Pichia pastoris strains were transferred to 10 ml of BMMY liquid medium and cultured at 30℃ and 280 rpm for 120 h with shaking. 200 μl of each bacterial culture was taken, centrifuged at 6000 rpm for 5 min, the precipitate was discarded, and 50 μl of the supernatant was added to 10 μl of 6× protein loading buffer. The mixture was boiled at 100℃ for 10 min, and the samples were taken for SDS-PAGE electrophoresis.

[0133] Ten single colonies of recombinant Pichia pastoris strain X33 were used as negative controls, with strain X33 transformed with pPICZαA vector (i.e., pPICZαA / X33); ten single colonies of recombinant Pichia pastoris strain GS115 were used as negative controls, with strain GS115 transformed with pPIC9k vector (i.e., pPIC9k / GS115); ten single colonies of recombinant Pichia pastoris strain KM71 were used as negative controls, with strain KM71 transformed with pPIC9k vector (i.e., pPIC9k / KM71); and ten single colonies of recombinant Pichia pastoris strain SMD1168 were used as negative controls, with strain SMD1168 transformed with pPIC9k vector (i.e., pPIC9k / SMD1168).

[0134] like Figure 10As shown, compared to pPICZαA / X33, among the 10 single colonies, 5 single colonies (pPICZαA-CollagenⅠ / X33-1 to pPICZαA-CollagenⅠ / X33-3, pPICZαA-CollagenⅠ / X33-7, and pPICZαA-CollagenⅠ / X33-10) showed a distinct, darker protein band between 80 kDa and 115 kDa, indicating relative stability compared to other constructed strains. The remaining 5 single colonies showed a lighter protein band between 80 kDa and 115 kDa, all consistent with the protein bands in the CollagenⅠ / X33-10 strain. The expected size of the agen I protein was consistent, but most of them had been degraded into smaller proteins. In addition, during the screening of expression in 10 single colonies, a Collagen I protein band appeared between 80kDa and 115kDa. Since the Collagen I protein of this invention is a secreted protein, on the one hand, the pure natural type I collagen itself has unstable properties, and on the other hand, the culture density during the screening of recombinant colonies is relatively high. In addition to Collagen I protein, there are also a small amount of protease secreted by Pichia pastoris (or released due to rupture) in the supernatant of the bacterial solution, which will lead to partial degradation of Collagen I protein.

[0135] The other three recombinant Pichia pastoris strains also showed similar identification results.

[0136] Compared to pPIC9k / GS115, 10 single colonies (pPIC9k-CollagenⅠ / GS115-1 to pPIC9k-CollagenⅠ / GS115-10) showed a distinct protein band between 80kDa and 115kDa, consistent with the expected size of CollagenⅠ protein, and also showed a degradation band of CollagenⅠ protein between 50kDa and 65kDa.

[0137] Compared with pPIC9k / KM71, 10 single colonies (pPIC9k-CollagenⅠ / KM71-1 to pPIC9k-CollagenⅠ / KM71-10) showed a distinct protein band between 80 and 115 kDa, consistent with the expected size of CollagenⅠ protein, and also showed a degradation band of CollagenⅠ protein between 50 and 65 kDa.

[0138] Compared with pPIC9k / SMD1168, 10 single colonies (pPIC9k-CollagenⅠ / SMD1168-1 to pPIC9k-CollagenⅠ / SMD1168-10) showed a distinct protein band between 80kDa and 115kDa, consistent with the expected size of CollagenⅠ protein, and also showed a degradation band of CollagenⅠ protein between 50kDa and 65kDa.

[0139] The above results indicate that the corresponding recombinant plasmids were successfully transformed into all four recombinant Pichia pastoris strains, and the recombinant type I collagen of the present invention was successfully expressed and secreted into the culture medium.

[0140] In summary, compared with the other three recombinant Pichia pastoris strains, the overall Collagen I protein expression level of the recombinant Pichia pastoris X33 strain was higher, by nearly 50%, and the Collagen I protein expression level of the 10th single colony (i.e., pPICZαA-CollagenⅠ / X33-10) was the highest and most stable. Therefore, it was used for the subsequent preparation of recombinant type I collagen.

[0141] Twenty-four tubes of pPICZαA-CollagenⅠ / X33-10 bacterial culture were aliquoted and stored, designated as generation F0. A series of tests were performed on the F0 generation pPICZαA-CollagenⅠ / X33-10. The results showed that when the recombinant strain was streaked onto YPD solid medium containing 200 μg / ml bleomycin, the colonies were large, thick, smooth, moist, mostly milky white, and had an alcoholic aroma, consistent with yeast culture characteristics. Under a standard optical microscope, the recombinant strain appeared round, oval, or sausage-shaped. PCR identification confirmed that the recombinant strain could amplify the target fragment of 2583 bp.

[0142] Example 4: Preparation process of recombinant type I collagen

[0143] 1. pPICZαA-CollagenⅠ / X33-10 fermentation

[0144] pPICZαA-CollagenⅠ / X33-10 was inoculated at a rate of 0.1% v / v into a 500ml reaction tube containing 100ml of BMGY liquid medium for amplification culture.

[0145] OD of the bacterial culture 600 When the OD value reaches 5.0, change the solution to remove the OD. 600 The bacterial culture of 1.0 was inoculated into 100 ml of BMMY liquid medium containing 2% w / v sorbitol for induction expression. The induction period was 72 h, and then the fermentation was terminated.

[0146] Fermentation broth samples were taken at 24h, 48h, and 72h after induction, and the expression level of Collagen I protein was detected by SDS-PAGE electrophoresis. Figure 11 As shown, the expression level of Collagen I protein gradually increased with the extension of induction time, and there was no degradation band of Collagen I protein. This is because the Collagen I protein expressed by pPICZαA-CollagenⅠ / X33-10 has good stability, so it is not degraded by non-inherent factors such as cell rupture caused by excessive density of fermentation strains.

[0147] 2. Protein purification

[0148] The fermentation broth was collected, centrifuged, the precipitate was removed, the supernatant was collected, and purified using a strong cation exchange chromatography column (SP column).

[0149] Equilibrate the SP column to a stable conductivity and pH using 5CV–10CV Buffer B (25mM acetate-sodium acetate buffer (NaAc-HAc), pH 4.0); mix the fermentation broth supernatant with Buffer A (0.5% v / v acetic acid) at a volume ratio of 1:1, load the mixture onto the SP column, and collect the flow-through in a clean container, denoted as FT.

[0150] After loading the sample, rinse first with 5CV Buffer B, then with 5CV Buffer C (25mM NaAC-HAC, pH 5.5), and then with 5CV Buffer D (20mM PB, pH 6.0). Collect the samples rinsed with the three buffers into clean containers and label them as EpH 4.0, EpH 5.5, and EpH 6.0, respectively.

[0151] After rinsing, the target protein was eluted with Buffer E (20mM PB, 50mM NaCl, pH 6.5). The elution products were collected in sterile containers, and the UV absorbance of the elution products at A280 nm was monitored in real time. The elution products corresponding to the first protein peak were collected. A total of two elution products were collected, with collection volumes of 15 mL and 27.5 mL respectively, and were labeled as E50-1 and E50-2 respectively.

[0152] The collected FT, EpH4.0, EpH5.5, EpH6.0, E50-1, and E50-2 were analyzed by SDS-PAGE electrophoresis.

[0153] like Figure 12As shown, lane 1 (supernatant diluted 4 times with 0.5% acetic acid), lane 6 (E50-1), and lane 7 (E50-2) all showed target protein bands at 80-115 kDa, indicating that the recombinant type I collagen was successfully purified.

[0154] Subsequently, E50-1 and E50-2 were merged to obtain the combined purified product of recombinant type I collagen.

[0155] 3. Dialysis and lyophilization

[0156] The combined purified product of recombinant type I collagen was dialyzed using a dialysis bag with buffer (20 mM PB, 50 mM NaCl, pH 6.5) and a MW3500 (i.e., a molecular weight cutoff of 3.5 kDa). After dialysis for 24 h, the dialysate was collected and lyophilized to obtain a lyophilized sample of recombinant type I collagen, which was a white solid powder.

[0157] Stability evaluation of recombinant type I collagen in test cases

[0158] 1. Evaluation Methods

[0159] The liquid samples (i.e. dialysate) of recombinant type I collagen obtained in Example 3 were placed into sealed tubes, sealed, and stored at 4°C. SDS-PAGE electrophoresis was performed on samples taken at 0, 1, 4, and 6 weeks of storage to analyze the degradation of recombinant type I collagen.

[0160] 2. Evaluation Results

[0161] like Figure 13 As shown, at storage times of 0, 1, 4, and 6 weeks, the liquid samples of recombinant type I collagen all exhibited a distinct protein band between 80 kDa and 115 kDa, with comparable band brightness and no extraneous or dragging bands. This indicates that the recombinant type I collagen prepared by this invention possesses extremely high stability and did not degrade during storage at 4°C for 6 weeks.

[0162] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

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

6.

2. A nucleic acid molecule, characterized in that, The recombinant type I collagen as described in claim 1 is encoded.

3. A biomaterial, characterized in that, It comprises the nucleic acid molecule of claim 2; the biological material is recombinant DNA, expression cassette, transposon, vector, cell or microorganism.

4. The use of the recombinant type I collagen of claim 1, the nucleic acid molecule of claim 2, or the biomaterial of claim 3 in the preparation of drugs, medical biomaterials, or medical devices.

5. A product containing the recombinant type I collagen as described in claim 1, characterized in that, The products mentioned are pharmaceuticals, medical biomaterials, or medical devices.

6. A method for preparing recombinant type I collagen, characterized in that, include: The microorganisms described in claim 3 are cultured, and the recombinant type I collagen is recovered from the culture product.

7. The preparation method according to claim 6, characterized in that, The microorganisms include yeast.

8. The preparation method according to claim 6 or 7, characterized in that, Also includes: The recovered recombinant type I collagen is purified by a method selected from one or more of affinity chromatography, ion exchange, hydrophobic chromatography, and salting out.

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