Hydroxyproline-modified high-thermal-stability recombinant human type i collagen and preparation method and application thereof

Abstract

The application discloses a kind of hydroxyproline modified high thermal stability recombinant I type humanized collagen and its preparation method and application, belong to the field of bioengineering technology.The hydroxyproline modified degradation-resistant recombinant I type humanized collagen C010001 prepared in the application has amino acid sequence selected from natural I type human collagen amino acid sequence, and has very high degradation resistance, and is hardly degraded in 80 DEG C environment for 4 hours.Compared with I type human collagen, it has better cell adhesion effect.The recombinant I type humanized collagen using the purification scheme of the application does not contain additional non-human amino acid residues, and the possibility of immune response in human body is lower, and the preparation method is simple, and the cost is low.

Description

Technical Field

[0001] This invention relates to a hydroxyproline-modified, highly thermally stable recombinant type I humanized collagen, its preparation method, and its application, belonging to the field of bioengineering technology. Background Technology

[0002] Collagen (COL) is the most abundant protein in the human body, accounting for approximately 25%-35% of total body protein. It is a large family with at least 28 different types (type I, type II, type III, etc.). Type I collagen is the most prevalent and common type, accounting for about 90% of all collagen. In nature, collagen is simply called collagen protein. It is a helical, fibrous functional protein composed of three polypeptide chains. Its characteristic triple helix sequence is GXY, where X and Y positions are often proline and hydroxyproline, respectively. Proline hydroxylation is the most common post-translational modification in collagen. The resulting product is trans-proline. 4 Hydroxyproline (Hyp) is crucial for the structural stability and function of collagen. Studies have shown that the lack of proline hydroxylation in collagen can destabilize the triple helix structure, thus affecting its binding with integrins.

[0003] Currently, collagen production processes are mainly divided into animal-derived extraction and recombinant processing. Animal-derived extraction primarily involves extracting from animal tissues (such as cattle, pigs, and fish). However, animal-derived extraction carries risks related to viruses and pathogens, immune rejection and allergic reactions, batch-to-batch quality instability, religious and ethical limitations, and chemical residues.

[0004] There are numerous reports on the preparation of recombinant collagen, mainly focusing on the expression of human collagen genes. For example, Chinese patent CN113621052B discloses a method for producing recombinant collagen by expressing a spliced ​​sequence of a human partial gene in *E. coli*. Chinese patent CN115991764B discloses a method for producing collagen by co-transforming a hydroxylase gene and a spliced ​​sequence of a human partial gene into *E. coli*. This method uses two independent plasmids to simultaneously transform into the host, resulting in a complex recombinant protein production process and limited yield. Chinese patents CN112626074B and CN109022464A utilize yeast to produce hydroxylated recombinant collagen, but the hydroxylation ratio of the product is not clearly determined. CN114853881A achieves a hydroxylated recombinant collagen with a 63% hydroxylation ratio by stepwise expression of hydroxylase and collagen, improving its thermal stability from approximately 30°C to approximately 35°C. In the production and sterilization processes of cosmetics and medical devices, raw materials are present in high-temperature (70°C) environments. High temperatures can denature and degrade protein raw materials, making them ineffective. Therefore, protein raw materials that do not degrade or precipitate under high temperatures are particularly important.

[0005] Current methods for producing recombinant collagen involve tandemly linking the affinity tag and enzyme cleavage site to the target recombinant protein in the carrier design. However, due to the specific spatial structures of both, steric hindrance can occur, leading to low cleavage efficiency or even complete cleavage failure. Furthermore, the purification process is cumbersome, requiring a second affinity chromatography step after the enzyme digestion to remove the tag. This process not only struggles to completely remove tag impurities through affinity chromatography, but also, when the target product has a similar molecular weight to the tag, any residues present greatly complicate subsequent identification, separation, and purification, potentially introducing hazardous substances and affecting the quality of the final product.

[0006] Therefore, a recombinant type I humanized collagen with a defined proline hydroxylation ratio, no additional non-human amino acid residues, high temperature resistance to degradation, strong bioactivity, and simple, readily available, low-cost, and controllable production process is particularly important. Summary of the Invention

[0007] To address the shortcomings of the existing technologies, this invention provides a hydroxyproline-modified, highly thermally stable recombinant type I humanized collagen, its preparation method, and its applications. The aim is to solve the technical problems of existing technologies, such as the lack of a defined proline hydroxylation ratio, absence of additional non-human amino acid residues, high-temperature degradation resistance, strong bioactivity, and the simple, readily available, and low-cost preparation method for producing humanized collagen.

[0008] The first technical solution provided by the present invention is a recombinant plasmid, wherein the recombinant plasmid uses pET28YZT plasmid as an expression vector and carries a proline hydroxylase encoding gene and a recombinant type I humanized collagen encoding gene containing an enzyme digestion sequence.

[0009] The pET28YZT plasmid has a second gene expression element inserted into the NcoI and XhoI multiple cloning sites of pET-28a(+). The second gene expression element contains an NcoI restriction site, a 6HIS tag, a BamHI restriction site, a HindIII restriction site, a T7 promoter, a lac operator, a ribosome aggregation site, an NdeI restriction site, and an XhoI restriction site. The nucleotide sequence of the second gene expression element is SEQ ID No. 1. The nucleotide sequence of the proline hydroxylase encoding gene is shown in SEQ ID No. 3, and the nucleotide sequence of the recombinant type I humanized collagen encoding gene containing the enzyme digestion sequence is shown in SEQ ID No. 7.

[0010] The second technical solution provided by the present invention is a recombinant cell, wherein the recombinant cell is transformed with the recombinant plasmid described in the first technical solution.

[0011] In some embodiments, the recombinant cells use Escherichia coli as the host cell.

[0012] The present invention provides a third technical solution, which is a method for hydroxylation of recombinant type I humanized collagen. The method involves inoculating the recombinant cells described in the second technical solution into a culture medium for culture and inducing expression.

[0013] In some embodiments, the collected cultured bacterial solution is subjected to cell disruption, affinity chromatography, enzyme digestion to remove the tag, ion exchange chromatography, concentration and liquid replacement, and purification of recombinant type I humanized collagen to obtain proline hydroxylation modification with a purity greater than >95%.

[0014] In some embodiments, the culture medium contains 5 g / L yeast extract, 10 g / L yeast peptone, and 5 g / L sodium chloride.

[0015] In some embodiments, the culture conditions are as follows: the inoculum is 1%, and the culture is carried out at 37°C and 180 rpm until the OD600 value is 0.6. Then, IPTG is added to a final concentration of 0.25 mmol / L for induction, and the culture is carried out at 16°C and 180 rpm for 18 hours.

[0016] The four technical solutions provided by this invention are a hydroxylated recombinant type I humanized collagen, which is obtained by the method described in the third technical solution.

[0017] The present invention provides four technical solutions, the first of which describes the recombinant plasmid, the second of which describes the recombinant cell, and the third of which describes the method, for the preparation of hydroxylated recombinant type I humanized collagen or products containing hydroxylated recombinant type I humanized collagen.

[0018] In some embodiments, the product includes cosmetics and pharmaceuticals.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: Through the implementation of the technical solution of this invention, the hydroxyproline-modified, degradation-resistant recombinant type I humanized collagen C010001 prepared by this invention has an amino acid sequence selected from the amino acid sequence of natural type I human collagen and exhibits extremely high degradation resistance, showing almost no degradation after 4 hours at 80°C. Compared with type I human collagen itself, it has a better cell adhesion effect. Using the purification method of this invention, the recombinant type I humanized collagen does not contain additional non-human amino acid residues, and the possibility of inducing an immune response when applied to humans is low. Moreover, the preparation method is simple and low in cost. Attached Figure Description

[0020] Figure 1The image shows the plasmid map of the recombinant expression plasmid pET28YZT-C010001, where the amino acid sequence corresponding to C010001 is SEQ ID No. 7.

[0021] Figure 2 The results of fermentation condition screening based on SDS-PAGE are shown. Lanes 1 and 5 are molecular weight markers. Lanes 2-4 are the fermentation results of hydroxyproline-modified recombinant type I humanized collagen (C010001) in liquid media M1, M2, and M3. Lanes 6-8 are the fermentation results of non-hydroxyproline-modified recombinant type I humanized collagen (C009001) in liquid media M1, M2, and M3.

[0022] Figure 3 The results of affinity tag removal efficiency detection based on SDS-PAGE are shown. Lane 1 is the molecular weight marker, lane 2 is the recombinant type I humanized collagen modified with hydroxyproline after 3C enzyme digestion, and lane 3 is the recombinant type I humanized collagen modified with undigested hydroxyproline.

[0023] Figure 4 The results are based on SDS-PAGE proline hydroxylation assays. Lane 1 is the molecular weight marker, lane 2 is non-proline hydroxylated recombinant type I humanized collagen, lane 3 is proline hydroxylated recombinant type I humanized collagen (batch 1), and lane 4 is proline hydroxylated recombinant type I humanized collagen (batch 2).

[0024] Figure 5 The results of the degradation resistance test of non-hydroxyproline modified recombinant type I humanized collagen (C009001) based on SDS-PAGE are shown. Lane 1 is the molecular weight marker, lane 2 is the untreated non-hydroxyproline modified recombinant type I humanized collagen, and lanes 3-10 are the non-hydroxyproline modified recombinant type I humanized collagen treated at 30℃-100℃.

[0025] Figure 6 The results of the SDS-PAGE degradation resistance test of hydroxyproline-modified recombinant type I humanized collagen (C010001) are shown. Lane 1 is the molecular weight marker, lane 2 is the untreated hydroxyproline-modified recombinant type I humanized collagen, and lanes 3-10 are the treated hydroxyproline-modified recombinant type I humanized collagen at 30℃-100℃.

[0026] Figure 7 To determine the cell adhesion activity assay results, the lactate dehydrogenase content of cells adhering to the bottom of the culture dish was measured using an LDH assay kit.

[0027] Figure 8The image shows the collagen electrophoresis pattern for Comparative Example 1, which compares the collagen expression of different carriers based on SDS-PAGE. Lane 1 is the molecular weight marker, lane 2 is the result of affinity chromatography for collagen using the pET DUET1 carrier, and lane 3 is the result of affinity chromatography for collagen using the pET28YZT carrier.

[0028] Figure 9 The image shows the collagen electrophoresis results for Comparative Example 2, which compares the collagen expression of different carriers based on SDS-PAGE. Lane 1 is the molecular weight marker, lane 2 is the result of affinity chromatography for collagen using the pET DUET1 carrier, and lane 3 is the result of affinity chromatography for collagen using the pET28YZT carrier. Detailed Implementation

[0029] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0030] Example 1: Preparation of hydroxyproline-modified, highly thermostable recombinant type I humanized collagen 1. Construction of pET28YZT plasmid A co-expression vector containing kanamycin resistance was modified from the commercial vector pET-28a(+), namely pET28YZT. The plasmid was constructed as follows: necessary components for the expression of a second gene were inserted into the NcoI and XhoI multiple cloning sites of pET-28a(+), namely, an NcoI restriction site, a 6HIS tag, a BamHI restriction site, a HindIII restriction site, a T7 promoter, a lac operator, a ribosome aggregation site, an NdeI restriction site, and an XhoI restriction site. Its nucleic acid sequence is SEQ ID No. 1. The two pairs of multiple cloning sites (BamHI-HindIII and NdeI-XhoI) enable the co-expression of two recombinant proteins. Both proteins have affinity purification tags at their N-terminus and C-terminus, respectively. The recombinant protein with the N-terminal tag can be removed by restriction enzyme digestion, thus enabling stepwise purification of the two proteins.

[0031] 2. Construction of recombinant plasmids and expression strains for recombinant type I humanized collagen: In this embodiment, taking Escherichia coli BL21 in a prokaryotic system as an example, the designed sequence was optimized according to codon preference, and the corresponding gene was synthesized by GenScript. The nucleic acid sequence of degradation-resistant type I human collagen (SEQ ID No. 7) with protease cleavage sites was ligated to the pET28YZT plasmid via the BamHI and HindIII multiple cloning sites to obtain expression plasmid C009001; then, the nucleic acid sequence of proline hydroxylase (SEQ ID No. 3) was ligated to plasmid C009001 via the NdeI and XhoI multiple cloning sites to obtain expression plasmid C010001, thus completing the construction of the expression vector.

[0032] 3. Expression and purification of recombinant type I humanized collagen protein: In this embodiment, the method for expressing and purifying recombinant human type I collagen includes: (1) Protein expression: Single colonies of recombinant plasmid C010001 transformed into Escherichia coli BL21 were plated and incubated overnight at 37°C and 180 rpm in 20 mL of LB liquid medium containing 100 μg / mL Kan. Then, 1% of each colony was inoculated into 1000 mL of M1, M2, and M3 liquid medium containing 100 μg / mL Kan, respectively, and incubated at 37°C and 180 rpm until the OD600 value reached 0.6. IPTG was then added to a final concentration of 0.25 mmol / L for induction, and the culture was carried out at 16°C and 180 rpm for 18 hours. The cells were collected by centrifugation at 4000 rpm and 4°C for 20 min. The composition of the M1, M2, and M3 liquid media is shown in Table 1.

[0033] Table 1 Element M1 M2 M3 Yeast extract 12g / L 16g / L 5g / L Yeast peptone 10g / L 10g / L 10g / L Sodium chloride 5g / L 5g / L 5g / L pH 7.4 7.4 7.4 (2) Cell disruption: Take 1L of the bacterial cells from (1) and resuspend them in 40mL Binding Buffer (20mM imidazole, 10mM Tris-HCl, 500mM NaCl, pH 7.4). Sonicate on ice (300W power, 2s disruption, 3s pause, 5min operation), repeating 3 times. Take 80μL of the disruption solution, add 20μL of 5× loading buffer, denature at 95℃ for 10min, and perform 12% SDS. PAGE electrophoresis analysis: After fragmentation, the sample was centrifuged at 12000 rpm for 5 min at 4℃. 80 μL of the supernatant was collected, and 20 μL of 5× loading buffer was added. The sample was then denatured at 95℃ for 10 min and subjected to 12% SDS-PAGE analysis. PAGE electrophoresis analysis.

[0034] The results are as follows Figure 2As shown, the expression of C010001 was as follows: in M1 medium, the target band was two bands, indicating that the collagen exists in a state where hydroxylation and non-hydroxylation coexist; in M2 medium, the target band was one band with a small molecular weight, indicating that the collagen is in a non-hydroxylated state; and in M3 medium, the target band was one band with a large molecular weight, indicating that the collagen is in a hydroxylated state. Plasmid C009001 was used as a control. C009001 showed a single band with a small molecular weight in all three media, indicating that the collagen was in a non-hydroxylated state, consistent with the design objective.

[0035] (3) Nickel column affinity chromatography: Gravity column is used, and nickel column is selected (Changzhou Tiandi Renhe Co., Ltd., product number: SA1500). The resuspension buffer consisted of 20 mM imidazole, 10 mM Tris-HCl, 500 mM NaCl, pH 7.4; the washing buffer consisted of 20 mM imidazole, 10 mM Tris-HCl, 500 mM NaCl, pH 7.4; the elution buffer consisted of 20 mM imidazole, 10 mM Tris-HCl, 500 mM NaCl, pH 7.4; finally, an appropriate amount of His-tagged 3C protease (Ingentop, ME0461) was added. After incubating the mixture at 4°C for 16 hours, samples were taken and identified by SDS-PAGE electrophoresis. The results are as follows. Figure 3 As shown, the recombinant purified tag of recombinant type I humanized collagen can be completely removed using the Inspiro 3C enzyme.

[0036] (4) Dialyze the enzyme-digested protein into solution A (20mM Tris, pH 7.4); equilibrate the HiSelect Smac Q 40 (Changzhou Tiandi Renhe Co., Ltd., catalog number: SI035C47) ion exchange column with 5 column volumes of solution A; filter the enzyme switching solution through a 0.22um syringe filter and pass the protein through the HiSelect Smac Q 40 column, and collect the flow-through liquid, which is the target protein from which the host protein has been removed.

[0037] (5) The purity of the obtained C010001 protein was determined by SDS-PAGE. The specific process was as follows: 40 μL of purified protein solution was taken and 10 μL of 5× protein loading buffer (250 mM Tris-HCl (pH: 6.8), 10% SDS, 0.5% bromophenol blue, 50% glycerol, 5% β-mercaptoethanol) was added to each well and boiled in 100℃ boiling water for 10 min. Then, 10 μL of the protein solution was added to each well in the SDS-PAGE protein gel and run at 120V for 50 min. The protein was then stained with Coomassie Brilliant Blue staining solution (0.1% Coomassie Brilliant Blue R-250, 25% isopropanol, 10% glacial acetic acid) for 20 min and then destained with protein destaining solution (10% acetic acid, 5% ethanol).

[0038] Example 2: Determination of the hydroxylation ratio of hydroxyproline-modified recombinant type I humanized collagen 1. To determine whether collagen is hydroxylated, two independent batches of proline-hydroxylated recombinant type I humanized collagen (C010001) and non-proline-hydroxylated recombinant type I humanized collagen (C009001), purified in Example 1, were analyzed by SDS-PAGE. Theoretically, the proline-hydroxylated recombinant type I humanized collagen has a higher molecular weight. Furthermore, the different hydroxylation ratios will result in different molecular weights.

[0039] The results are as follows Figure 4 As shown, the proline-hydroxylated recombinant type I humanized collagen (C010001) has a higher molecular weight than the non-proline-hydroxylated recombinant type I humanized collagen (C009001), proving that hydroxylation exists in the proline-hydroxylated recombinant type I humanized collagen (C010001). Two independent batches of proline-hydroxylated recombinant type I humanized collagen (C010001) have the same molecular weight, indicating small batch-to-batch differences in the produced proline-hydroxylated recombinant type I humanized collagen (C010001).

[0040] 2. To specifically determine the proline hydroxylation ratio of collagen, the samples were commissioned to the Guangdong Academy of Sciences Testing and Analysis Institute for amino acid analysis according to GB 5009.124-2016. Two batches of samples were independently tested.

[0041] The hydroxylation rate of proline is calculated as follows: Hydroxylation rate = hydroxyproline / (hydroxyproline + proline).

[0042] The test results showed that, according to the amino acid analysis of two independent batches of collagen, more than 40% of the proline in the proline hydroxylated recombinant type I humanized collagen (C010001) was hydroxylated proline, and the hydroxylation rate of the two batches was similar with small batch-to-batch differences.

[0043] Example 3: Evaluation of the degradation resistance of hydroxyproline-modified recombinant type I humanized collagen. To verify whether proline hydroxylation modification can enhance the degradation resistance of type I collagen, proline-hydroxylated recombinant type I humanized collagen (C010001) and non-proline-hydroxylated recombinant type I humanized collagen (C009001) were incubated at 30℃, 40℃, 50℃, 60℃, 70℃, 80℃, 90℃, and 100℃ for 4 hours, respectively, to identify whether the recombinant proteins degraded. The specific implementation method includes the following steps: (1) Divide the collagen stock solution into 9 equal parts, and place one part in a refrigerator at 2-8℃ as a control.

[0044] (2) The water bath temperatures were 30℃, 40℃, 50℃, 60℃, 70℃, 80℃, 90℃ and 100℃ respectively. The remaining samples were placed in water baths at different temperatures and incubated for 4 hours.

[0045] (3) SDS-PAGE was used to determine whether collagen was degraded.

[0046] The results are as follows Figure 5 As shown, the main band of proline-hydroxylated recombinant type I humanized collagen (C009001) weakened significantly at temperatures above 70°C, indicating that the collagen underwent denaturation, precipitation, and degradation. However, after proline hydroxylation modification, a very small portion of the collagen in C010001 degraded at temperatures above 90°C (e.g., ...). Figure 6 This means that proline hydroxylation modified recombinant type I humanized collagen (C010001) has extremely high thermal stability.

[0047] Example 4: Evaluation of the biological activity of hydroxyproline-modified recombinant type I humanized collagen For methods to detect collagen activity, please refer to the literature Juming Yao, Satoshi Yanagisawa, Tetsuo Asakura, Design, Expression and Characterization of Collagen. LikeProteins Based on the Cell Adhesive and Crosslinking Sequences Derived fromNative Collagens,J Biochem.136,643 649 (2004). Lactate dehydrogenase (LDH) is a stable cytoplasmic enzyme that is widely present in various organisms, and the amount of this enzyme can linearly reflect the number of adherent cells.

[0048] The specific implementation method is as follows: (1) The concentration of the protein sample to be tested was detected by ultraviolet absorption method, including commercial human collagen (Sigma, C7774), proline hydroxylation modified recombinant type I human collagen (C010001) and non-proline hydroxylation modified recombinant type I human collagen (C009001) provided by the present invention.

[0049] (2) The specific method for detecting the concentration of the protein sample by ultraviolet absorption method can be described according to the method described in patent CN113621052B. Specifically, the ultraviolet light absorption of the sample at 215nm and 225nm is measured respectively, and the concentration is determined by the empirical formula C (μg / mL) = 144 × (A215) (A225) Calculate protein concentration, note that the test must be performed when A215 < 1.5. (Reference: Walker JM. The Protein Protocols Handbook, second edition. Humana Press. 43) 45.). After detecting the protein concentration, adjust the concentration of all proteins to be tested to 0.05 mg / ml using PBS.

[0050] (3) Add 10 to each hole 6 3T3 cells in good culture condition (from Hangzhou Qizhenhu Biotechnology Co., Ltd.) were incubated at 37°C in a 5% carbon dioxide environment for 60 minutes.

[0051] (4) Add 100 μL of various protein solutions and blank PBS solution to the 96-well plate and let it stand at room temperature for 60 min.

[0052] (5) Discard the culture medium and add 100 μL of 1% BSA-PBS blocking solution to each well for blocking. (6) Wash each well with PBS 3 times.

[0053] (7) After adding the LDH release reagent, mix thoroughly by pipetting and continue incubation. Use an LDH detection kit (Roche, 04744926001) to detect the absorbance at OD492 nm and OD690 nm in the supernatant. The absorbance difference between OD492 nm and OD690 nm represents the number of adherent cells. Based on the values ​​of the blank control, the cell adhesion rate can be calculated. Using commercially available human collagen (Sigma, C7774) as the positive well, the cell adhesion rate is calculated using the following formula: Cell adhesion rate = (Test wells) / (Test wells) / Test wells. (Blank well) × 100% / (Positive well) (blank hole).

[0054] The results are as follows Figure 7 As shown in the comparison, compared with commercially available human collagen (Sigma, C7774), the proline hydroxylation modified recombinant type I humanized collagen (C010001) and the non-proline hydroxylation modified recombinant type I humanized collagen (C009001) of the present invention both have excellent bioadhesion activity, and the proline hydroxylation modified recombinant type I humanized collagen (C010001) has even better bioadhesion activity.

[0055] Comparative Example 1 Replace the pET28YZT vector in Example 1 with the pET DUET1 vector, and keep other conditions or parameters the same as in Example 1.

[0056] The recombinant plasmid used pET DUET1 as the expression vector, carrying the proline hydroxylase encoding gene and the recombinant type I humanized collagen encoding gene containing restriction enzyme sequences. The nucleic acid sequence of degradation-resistant type I human collagen (SEQ ID No. 7) with protease cleavage sites was ligated to the pET DUET1 plasmid via BamHI and HindIII multiple cloning sites to obtain the expression plasmid C009001DUET; then, the nucleic acid sequence of proline hydroxylase (SEQ ID No. 3) was ligated to the C009001DUET plasmid via NdeI and XhoI multiple cloning sites to obtain the expression plasmid C010001DUET, completing the construction of the expression vector. Expression and purification were performed using *E. coli*, and the fermentation and purification parameters were exactly the same as in the previous example. Purity was detected using SDS-PAGE. Figure 8 As shown, after fermentation and affinity purification using the pET DUET1 vector, the purity and yield of C010001DUET were slightly lower than those of our C010001. This indicates that our vector construction method is more suitable for our product.

[0057] Figure 8 To compare collagen expression using different SDS-PAGE vectors, lane 1 is the molecular weight marker, lane 2 is the result of affinity chromatography of collagen using the pET DUET1 vector, and lane 3 is the result of affinity chromatography of collagen using the pET28YZT vector.

[0058] Comparative Example 2 After using the MBP tag, the recombinant protein needs to have the tag removed, otherwise an immune response will definitely occur. After digestion with the recombinant protein, the product is the target protein and the MBP tag. The MBP tag has a molecular weight of 40-42 kDa, while the product in Example 1 has a molecular weight of 20-35 kDa. Therefore, after digestion, dialysis buffer needs to be added before passing the digested product through an MBP chromatography column to remove the MBP tag. However, there are several problems: (1) The added or removed steps are not suitable for industrial production, while 6HIS does not require this step; (2) Passing the product through an MBP chromatography column again cannot guarantee that the MBP tag will be completely removed. At the same time, if a small amount of MBP tag is mixed in the product, it cannot be removed and detected in subsequent steps, increasing the product risk.

[0059] Therefore, co-expression plasmids C009001MBP and C010001MBP with MBP tags were constructed in this comparative example. C009001MBP was used for the production of non-proline-hydroxylated collagen, and C010001MBP was used for the production of proline-hydroxylated collagen. The vector construction process involved inserting an MBP tag sequence between NcoI and BamH in C009001 and C01001. The amino acid sequence of the MBP tag is shown in SEQ ID No. 10, and the nucleotide sequence of the gene encoding the MBP tag is shown in SEQ ID No. 11. C009001MBP and C010001MBP were transformed into E. coli for fermentation and the proteins were purified according to a commercial MBP purification kit. The results are as follows: Figure 9 As shown, C009001MBP and C010001MBP have the same molecular weight, indicating that MBP affects the proline hydroxylation of collagen.

[0060] Figure 9 To compare collagen expression using different SDS-PAGE vectors, lane 1 is the molecular weight marker, lane 2 is the result of affinity chromatography of collagen using the pET DUET1 vector, and lane 3 is the result of affinity chromatography of collagen using the pET28YZT vector.

[0061] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A recombinant plasmid, characterized in that, The recombinant plasmid uses pET28YZT plasmid as an expression vector and carries a proline hydroxylase encoding gene and a recombinant type I humanized collagen encoding gene containing enzyme digestion sequences. The pET28YZT plasmid has a second gene expression element inserted into the NcoI and XhoI multiple cloning sites of pET-28a(+). The second gene expression element contains an NcoI restriction site, a 6HIS tag, a BamHI restriction site, a HindIII restriction site, a T7 promoter, a lac operator, a ribosome aggregation site, an NdeI restriction site, and an XhoI restriction site. The nucleotide sequence of the second gene expression element is SEQ ID No.

1. The nucleotide sequence of the proline hydroxylase encoding gene is shown in SEQ ID No. 3, and the nucleotide sequence of the recombinant type I humanized collagen encoding gene containing the enzyme digestion sequence is shown in SEQ ID No.

7.

2. A recombinant cell, characterized in that, The recombinant cell transformation includes the recombinant plasmid as described in claim 1.

3. The recombinant cell according to claim 2, characterized in that, The recombinant cells used Escherichia coli as the host cell.

4. A method for hydroxylation of recombinant type I humanized collagen, characterized in that, The method involves inoculating the recombinant cells according to any one of claims 2 to 3 into a culture medium for culture and inducing expression.

5. The hydroxylation method according to claim 4, characterized in that, The collected cultured bacterial solution was subjected to cell disruption, affinity chromatography, enzyme digestion to remove the tag, ion exchange chromatography, concentration and liquid replacement, and purification of recombinant type I humanized collagen to obtain proline hydroxylation modification with a purity greater than >95%.

6. The hydroxylation method according to claim 4, characterized in that, The culture medium contains 5 g / L yeast extract, 10 g / L yeast peptone and 5 g / L sodium chloride.

7. The hydroxylation method according to claim 4, characterized in that, The culture conditions were as follows: the inoculum was 1%, and the culture was carried out at 37°C and 180 rpm until the OD600 value was 0.

6. Then, IPTG was added to a final concentration of 0.25 mmol / L for induction, and the culture was carried out at 16°C and 180 rpm for 18 hours.

8. A hydroxylated recombinant type I humanized collagen, characterized in that, The hydroxylated recombinant type I humanized collagen is obtained by the method described in any one of claims 4 to 7.

9. The use of the recombinant plasmid of claim 1, the recombinant cell of any one of claims 2-3, or the method of any one of claims 4-7 in the preparation of hydroxylated recombinant type I humanized collagen or products containing hydroxylated recombinant type I humanized collagen.

10. The application according to claim 9, characterized in that, The products include cosmetics and pharmaceuticals.

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