Preparation method of CHO cell-expressed triple-helix structure type III recombinant human collagen and application thereof

Abstract

The application discloses a preparation method of CHO cell-expressed triple-helix structure type III recombinant human collagen and application thereof, and relates to the field of biotechnology. The preparation method comprises the following steps: obtaining the triple-helix structure type III recombinant human collagen through fermentation and purification of a recombinant engineering cell; and the construction method of the recombinant engineering cell comprises the following steps: transforming a host cell with a recombinant plasmid for expressing alpha 1 (III) and a recombinant plasmid for expressing hP4H alpha 1 and hP4HB to obtain the recombinant engineering cell. The preparation method can be used to prepare the type III recombinant human collagen with the triple-helix structure and a high proline hydroxylation rate, and the type III recombinant human collagen can be widely applied to the fields of cosmetics, medical cosmetology, medical devices, biomedical materials and the like, and has important market application value.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a preparation method of CHO cell-expressed triple-helical structure type III recombinant human collagen and application thereof. BACKGROUND

[0002] Collagen III is the main collagen in human skin, fascia and tendon. Collagen III is relatively small and exists between the epidermis and the dermis, which is called "baby collagen". The micro-collagen layer (Cushion Network) is mainly composed of collagen III and refers to the structure between the epidermis and the dermis, which is the key to supporting the epidermis and the first step of skin collapse. Collagen III is a typical collagen in the human body, which is composed of three alpha 1 peptide chains, each of which contains an amino-terminal peptide region, a characteristic (G-X-Y) n triple repeat sequence region and a carboxy-terminal peptide region. As an important natural biological protein, collagen has good biocompatibility, biological activity and degradability, and other unique functional characteristics, and can be widely used in many fields such as chemical industry, medicine, food and cosmetics. In particular, it is suitable for preparing various biological devices and is the most ideal source of biological materials, which has broad application prospects.

[0003] The collagen extract on the market is mainly obtained by treating animal tissues with acid, alkali and enzymatic hydrolysis. The processing process will destroy the natural structure of collagen, cause severe degradation and loss of biological activity. The extracted collagen peptides have different lengths, uneven properties, unstable quality and safety hazards of mad cow disease, foot-and-mouth disease and other viral infections. At the same time, the amino acid sequences of animal sources and human collagen are quite different, which belong to heterologous proteins and can cause immune rejection and allergic symptoms.

[0004] The production of recombinant collagen by genetic engineering technology can effectively avoid these defects. In the existing expression method of recombinant collagen, the prokaryotic (Escherichia coli) expression system and the Pichia pastoris expression system are mainly used to express human collagen in large-scale industrial production. There is no post-translational modification of proteins in Escherichia coli, and large-scale expression is intracellular expression, which needs to be lysed. A large amount of impurities of host proteins and natural endotoxins and peptidoglycans with cell wall components need to be removed by complex purification process. The Pichia pastoris expression system can perform certain post-translational modification of translated proteins, but compared with mammalian cells, the collagen expressed by yeast may have some folding errors, which need more steps to remove impurities and correct the wrong structure in the subsequent purification and quality control process, increasing the production cost and time.

[0005] CHO-K1 cell is Chinese hamster ovary cell, which has complete eukaryotic organelles and can perform post-translational modification of translated proteins closest to the natural state, thereby providing strong support for the realization of the biological function of proteins. The cell strain stably expressing exogenous genes established by CHO-K1 cell can be used for high-density fermentation production, and has the advantages of high expression and the like suitable for large-scale industrial production. The product can be secreted into the extracellular space, which can avoid impurity proteins caused by bacterial lysis, and the secreted product does not contain endotoxin and peptidoglycan. The genetic background of CHO-K1 cell is clear, and a variety of antibody drugs and vaccines have been successfully put on the market, and the corresponding product regulatory approval process is also clear and definite. At present, the technical research on the expression of triple helix structure type III recombinant human collagen by CHO-K1 cell is still relatively rare, therefore, it is extremely necessary to develop a method for efficiently expressing type III recombinant human collagen with triple helix structure by CHO cell. SUMMARY

[0006] The purpose of the present application is to provide a preparation method of CHO cell expressing triple helix structure type III recombinant human collagen and application thereof, so as to solve the problems existing in the prior art. The preparation method can be used to prepare type III recombinant human collagen with triple helix structure and high proline hydroxylation rate, which can be widely used in the fields of cosmetics, medical cosmetology, medical devices, biomedical materials and the like, and has important market application value.

[0007] In order to achieve the above purpose, the present application provides the following scheme:

[0008] The present application provides a construction method of recombinant engineering cell expressing triple helix structure type III recombinant human collagen, comprising the following steps: transforming a recombinant plasmid expressing alpha 1(III) and a recombinant plasmid expressing hP4H alpha 1 and hP4HB into a host cell to construct the recombinant engineering cell.

[0009] The amino acid sequences of alpha 1(III), hP4H alpha 1 and hP4HB are shown in SEQ ID NO. 1-3, respectively.

[0010] The host cell is CHO-K1 cell.

[0011] Further, the recombinant plasmid expressing alpha 1(III) comprises alpha 1(III) coding gene, and the nucleotide sequence of the alpha 1(III) coding gene is shown in SEQ ID NO. 4.

[0012] Further, the recombinant plasmid expressing alpha 1(III) is constructed by connecting the alpha 1(III) coding gene into an expression vector PEE12.4.

[0013] Further, the recombinant plasmid for expressing hP4Hα1 and hP4HB comprises a hP4Hα1 coding gene and a hP4HB coding gene;

[0014] The nucleotide sequence of the hP4Hα1 coding gene is shown as SEQ ID NO. 5.

[0015] The nucleotide sequence of the hP4HB coding gene is shown as SEQ ID NO. 6.

[0016] Further, the construction method of the recombinant plasmid for expressing hP4Hα1 and hP4HB comprises the following steps:

[0017] The hP4Hα1 coding gene is connected into the PEE6.4 expression vector carrying an antibiotic screening marker to construct a recombinant plasmid PEE12.4-Puro-hP4Hα1.

[0018] The hP4HB coding gene is connected into the expression vector PEE6.4 to construct a recombinant plasmid PEE6.4-hP4HB.

[0019] After the recombinant plasmid PEE12.4-Puro-hP4Hα1 and the recombinant plasmid PEE6.4-hP4HB are double-digested with Not I and BamH I, the connection is performed to obtain the recombinant plasmid for expressing hP4Hα1 and hP4HB.

[0020] The application further provides a recombinant engineering cell for expressing a triple helix structure III type recombinant human collagen, which is constructed according to the above construction method.

[0021] The application further provides an application of the above recombinant engineering cell in preparing a triple helix structure III type recombinant human collagen.

[0022] The application further provides a preparation method of a CHO cell for expressing a triple helix structure III type recombinant human collagen, which comprises the step of fermenting and purifying the triple helix structure III type recombinant human collagen by using the above recombinant engineering cell.

[0023] The application further provides a triple helix structure III type recombinant human collagen, which is prepared according to the above preparation method.

[0024] The application further provides an application of the above triple helix structure III type recombinant human collagen in preparing a functional biological material, a cosmetic or a medical device.

[0025] The application discloses the following technical effects:

[0026] The application provides a preparation method of CHO cell-expressed triple helix structure type III recombinant human collagen, and the preparation method can be used to prepare the type III recombinant human collagen with high proline hydroxylation rate and triple helix structure, so that the type III recombinant human collagen has good biomechanical properties and biocompatibility. The triple helix structure type III recombinant human collagen prepared by the application can be widely applied to the fields of cosmetics, medical cosmetology, medical devices, biomedical materials and the like, and has important market application value. BRIEF DESCRIPTION OF DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative effort on the basis of these drawings.

[0028] Figure 1 is a map of recombinant plasmid PEE12.4-α1(III);

[0029] Figure 2 is a map of recombinant plasmid PEE12.4-Puro-hP4Hα1;

[0030] Figure 3 is a map of recombinant plasmid PEE6.4-hP4HB;

[0031] Figure 4 is a map of recombinant plasmid PEE12.4-Puro-hP4Hα1-hP4HB;

[0032] Figure 5 is a Western Blot detection result map of α1(III) expression supernatant;

[0033] Figure 6 is a Western Blot detection result map of recombinant hP4Hα1 and hP4HB protein expression;

[0034] Figure 7 is a circular dichroism detection result map;

[0035] Figure 8 is a transmission electron microscope detection result map. DETAILED DESCRIPTION

[0036] The various exemplary embodiments of the present application will now be described in detail, which should not be considered as limiting the present application, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present application.

[0037] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Additionally, for a range of values of, for example, a parameter, an individual value or subrange within that range is also specifically disclosed. Each of the smaller ranges is also individually and specifically disclosed. The upper and lower limits of these smaller ranges can independently be included or excluded in the range, and are also endpoints of the range, subject to any specifically excluded endpoint.

[0038] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe in detail the methods and / or materials which are related to the present application. In the case of conflict between the present specification and any document incorporated herein by reference, the present specification will control.

[0039] Many modifications and variations of the present application described in the specific embodiments of the application can be made by those skilled in the art without departing from the spirit or scope of the application. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application. The specification and examples are illustrative only.

[0040] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean including, but not limited to.

[0041] Example 1

[0042] 1. Sequence synthesis

[0043] Synthesis of DNA sequence encoding α1(III), hP4Hα1, hP4HB.

[0044] The amino acid sequence of human collagen type III α1 chain (α1(III) chain) is shown in SEQ ID NO. 1;

[0045] The amino acid sequence of human proline hydroxylase hP4Hα1 is shown in SEQ ID NO. 2;

[0046] The amino acid sequence of human proline hydroxylase hP4HB is shown in SEQ ID NO. 3.

[0047] SEQ ID NO. 1:

[0048]

[0049] SEQ ID NO. 2:

[0050] MIWYILIIGILLPQSLAHPGFFTSIGQMTDLIHTEKDLVTSLKDYIKAEEDKLEQIKKWAEKLDRLTSTATKDPEGFVGHPVNAFKLMKRLNTEWSELENLVLKDMSDGFISNLTIQRQYFPNDEDQVGAAKALLRLQDTYNLDTDTISKGNLPGVKHKSFLTAEDCFELGKVAYTEADYYHTELWMEQALRQLDEGEISTIDKVSVLDYLSYAVYQQGDLDKALLLTKKLLELDPEHQRANGNLKYFEYIMAKEKDVNKSASDDQSDQKTTPKKKGVAVDYLPERQKYEMLCRGEGIKMTPRRQKKLFCRYHDGNRNPKFILAPAKQEDEWDKPRIIRFHDIISDAEIEIVKDLAKPRLRRATISNPITGDLETVHYRISKSAWLSGYENPVVSRINMRIQDLTGLDVSTAEELQVANYGVGGQYEPHFDFARKDEPDAFKELGTGNRIATWLFYMSDVSAGGATVFPEVGASVWPKKGTAVFWYNLFASGEGDYSTRHAACPVLVGNKWVSNKWLHERGQEFRRPCTLSELE.

[0051] SEQ ID NO. 3:

[0052] MLRRALLCLAVAALVRADAPEEEDHVLVLRKSNFAEALAAHKYLLVEFYAPWCGHCKALAPEYAKAAGKLKAEGSEIRLAKVDATEESDLAQQYGVRGYPTIKFFRNGDTASPKEYTAGREADDIVNWLKKRTGPAATTLPDGAAAESLVESSEVAVIGFFKDVESDSAKQFLQAAEAIDDIPFGITSNSDVFSKYQLDKDGVVLFKKFDEGRNNFEGEVTKENLLDFIKHNQLPLVIEFTEQTAPKIFGGEIKTHILLFLPKSVSDYDGKLSNFKTAAESFKGKILFIFIDSDHTDNQRILEFFGLKKEECPAVRLITLEEEMTKYKPESEELTAERITEFCHRFLEGKIKPHLMSQELPEDWDKQPVKVLVGKNFEDVAFDEKKNVFVEFYAPWCGHCKQLAPIWDKLGETYKDHENIVIAKMDSTANEVEAVKVHSFPTLKFFPASADRTVIDYNGERTLDGFKKFLESGGQDGAGDDDDLEDLEEAEEPDMEEDDDQKAVKDEL.

[0053] The optimized gene sequence of the α1(III) chain (SEQ ID NO. 1) is shown in SEQ ID NO. 4; the optimized gene sequence of the hP4Hα1 (SEQ ID NO. 2) is shown in SEQ ID NO. 5; and the optimized gene sequence of the hP4HB (SEQ ID NO. 3) is shown in SEQ ID NO. 6.

[0054] 2. Construction of the recombinant expression vector

[0055] The α1(III) coding gene sequence (SEQ ID NO. 4) was ligated into the expression vector PEE12.4, the hP4Hα1 coding gene sequence (SEQ ID NO. 5) was ligated into the expression vector PEE12.4-Puro (PEE12.4-Puro was constructed by inserting the puro gene into the pEE12.4 vector to replace GS (glutamine synthetase) as a selection marker), and the hP4HB coding gene sequence (SEQ ID NO. 6) was ligated into the expression vector PEE6.4, to construct the recombinant plasmid PEE12.4-α1(III) expressing α1(III), the recombinant plasmid PEE12.4-Puro-hP4Hα1 expressing hP4Hα1, and the recombinant plasmid PEE6.4-hP4HB expressing hP4HB, respectively. The recombinant plasmids PEE12.4-α1(III), PEE12.4-Puro-hP4Hα1, and PEE6.4-hP4HB were transformed into competent E. coli DH5α, respectively, and positive clones were selected on LB resistance plates containing Amp, and the recombinant plasmids were extracted and sequenced to confirm the correctness. The relevant plasmid maps are shown in Figures 1-3. Figures 1-3 .

[0056] Three μg of each of the recombinant plasmids PEE12.4-Puro-hP4Hα1 and PEE6.4-hP4HB were digested with Not I and BamH I at 37°C for 1 h, and recovered using a SanPrep column DNA gel recovery kit. The recovered two bands were ligated using T4 DNA Ligase. After ligation, the DH5α competent strain (purchased from Shanghai Shengong Bioengineering) was transformed, and positive clones were selected on LB resistance plates containing Amp, and the recombinant plasmid was extracted and sequenced to confirm the correctness, to obtain the recombinant plasmid PEE12.4-Puro-hP4Hα1-hP4HB. The relevant plasmid map is shown in Figure 4. Figure 4 .

[0057] 3. Recombinant α1(III) cell pool construction

[0058] 50 μg of recombinant plasmid PEE12.4-α1(Ⅲ) was linearized by digestion with PvuI enzyme at 37℃ for 1 h, and then the linearized plasmid was recovered using a PCR product purification kit. The linearized plasmid was electroporated into empty host CHO-K1 cells. The electroporated cells were transferred to T175 culture flasks, culture medium was added, and the flasks were incubated at 37℃ with 5% CO2 for 48 h. After 48 hours, the cells were gently aspirated from the culture flasks, centrifuged to remove the supernatant, and resuspended in pressurized medium (basal medium + 25 μM L-methionine sulfoxide imide). The cell density was adjusted to 80,000 cells / 600 μL. The cell suspension was seeded into 24-well plates and incubated at 37℃ with 5% CO2. The medium was changed every 7 days. At the 4th medium change, the cell supernatant from each well was collected for ELISA analysis, and the 10 cell pools with the best growth were selected. Ten cell lines were cultured in a fed-batch manner for screening. The viable cell density and viability were recorded daily. On the seventh day, the supernatant was harvested for ELISA analysis. The three cell pools with the highest OD were selected.

[0059] 4. Construction of recombinant α1(Ⅲ) monoclonal cell lines

[0060] The cells were cultured to the logarithmic growth phase and seeded at 0.8 cells / well / 200 μL. Single-clone wells were marked under a microscope and cultured for 14 days. Single-clone cells were then removed from the wells and transferred to 24-well plates for expansion culture. ELISA was performed after the cells had reached confluence with the bottom of the wells. Cell lines with high OD values ​​were selected for further expansion, while poorly growing cell lines were discarded. Twenty-one cell lines were subjected to fed-batch culture for selection. Cell density and viability were recorded daily. The supernatant was harvested on day seven and analyzed by Western blotting. The Western blotting results of the expression supernatant are shown below. Figure 5 As shown, fourteen monoclonal cell lines, 1E09, 1C07, 1E10, 1D06, 2F09, 1B01, 1H06, 2F04, 1D03, 2G03, 2F08, 1B08, 1C05, and 1A09, efficiently secreted the full-length α1(Ⅲ) sequence.

[0061] 5. Construction of recombinant hP4Hα1-hP4HB cell pool

[0062] Take 50 μg of recombinant plasmid PEE12.4-Puro-hP4Hα1-hP4HB, digest it with PvuI enzyme at 37℃ for 1 h to linearize it, and then use a PCR product purification kit to recover the linearized plasmid.

[0063] The linearized plasmid was electroporated into the 1H06 monoclonal cell strain screened from the above 4, and the electroporated cells were transferred into a T175 culture flask, and the culture medium was added and placed in a 37°C, 5% carbon dioxide incubator for 48 hours. After 48 hours, the cells in the culture flask were gently blown off, the supernatant was removed by centrifugation, and the pressure medium (basic medium + 30 μM L-methionine sulfoximine + 4 μg / mL Puromycin) was used for resuspension, and the cell density was adjusted to 80000 / 600 μL. The cell suspension was inoculated into a 24-well plate and placed in a 37°C, 5% carbon dioxide incubator for incubation. The cells were changed every 2 days, and the cell viability was detected every day. When the cell viability recovered and the 24-well plate was fully grown, the best 1 cell 15HB05 was taken for Western Blot detection, and the Western Blot detection result is shown in Figure 6 As can be seen from the results of Figure 6 , both hP4Hα1 and hP4HB can be detected in the cell lysate of 15HB05, indicating that 15HB05 successfully performed efficient secretion expression of the full-length sequence of hP4Hα1 and hP4HB proteins.

[0064] 6. Purification of recombinant α1(III)

[0065] According to the construction method of the monoclonal cell strain described in step 4 above, the monoclonal cell screening was performed on 15HB05 to obtain a high-yield monoclonal strain. The Fed-Batch fermentation culture was performed using a bioreactor, and after the end of the fermentation, the bioreactor fermentation liquid was collected, centrifuged at 4°C at high speed, and the precipitate was removed. The supernatant was again passed through a 0.45 μm filter membrane to keep the original liquid clear, and the purification treatment was performed:

[0066] Column loading: 500 mL NW ROSE TED FF, medium-pressure glass chromatography column size 50 / 400.

[0067] Column washing: 0.5M sodium hydroxide flushing 5 column volumes, flow rate 30 mL / min.

[0068] Equilibrium: 1×PBS equilibrium 15 column volumes, flow rate 30 mL / min.

[0069] Sample loading: 10L sample, flow rate 30 mL / min.

[0070] Equilibrium: 1×PBS equilibrium 5 column volumes, flow rate 30 mL / min.

[0071] Washing: 20mM imidazole flushing 2 column volumes, flow rate 30 mL / min, 1 bottle per column volume.

[0072] Elution: 250mM imidazole elution 5 column volumes, flow rate 30 mL / min, 1 bottle per column volume.

[0073] Column washing: Rinse with 0.5M sodium hydroxide for 5 column volumes at a flow rate of 30 mL / min.

[0074] Equilibration: Equilibrate with ddH2O for 15 column volumes at a flow rate of 30 mL / min.

[0075] Storage: Rinse with 20% ethanol for 3 column volumes, and finally store the packing material in 20% ethanol.

[0076] The eluent was subjected to electrophoresis to obtain a recombinant α1(Ⅲ) sample. After dialyzing into ddH2O at a suitable pH, the sample was lyophilized to obtain α1(Ⅲ) lyophilized powder.

[0077] 7. Structural characterization of α1(Ⅲ) lyophilized powder:

[0078] After reconstitution of α1(Ⅲ) lyophilized powder and acid hydrolysis, the peak area of ​​the target data was calculated by liquid chromatography-mass spectrometry (LC-MS) analysis and the identification results were obtained by standard curve method. The results showed that the hydroxyproline content reached more than 30% of the total proline.

[0079] The α1(Ⅲ) lyophilized powder was dissolved in water, and the results were analyzed using a circular dichroism spectrometer. The test results are as follows: Figure 7 As shown, it conforms to the circular dichroism spectral characteristics of triple-helix collagen.

[0080] Collagen samples were negatively stained and then examined using transmission electron microscopy. The test results are as follows: Figure 8 As shown, this illustrates the typical structural features of collagen microfibers with alternating light and dark areas.

[0081] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for constructing a recombinant engineered cell expressing a triple helical structure type III recombinant human collagen, characterized in that, The step of constructing the recombinant engineering cell comprises transforming a host cell with a recombinant plasmid expressing α1(III) and a recombinant plasmid expressing hP4Hα1 and hP4HB; The amino acid sequences of the α1(III), the hP4Hα1 and the hP4HB are shown in SEQ ID NO. 1-3, respectively; The host cell is a CHO-K1 cell; The recombinant plasmid expressing α1(III) comprises an α1(III) coding gene, and the nucleotide sequence of the α1(III) coding gene is shown in SEQ ID NO. 4; The recombinant plasmid expressing α1(III) is constructed by linking the α1(III) coding gene into an expression vector PEE12.4; The recombinant plasmid expressing hP4Hα1 and hP4HB comprises a hP4Hα1 coding gene and a hP4HB coding gene; The nucleotide sequence of the hP4Hα1 coding gene is shown in SEQ ID NO. 5; The nucleotide sequence of the hP4HB coding gene is shown in SEQ ID NO. 6; The construction method of the recombinant plasmid expressing hP4Hα1 and hP4HB comprises the following steps: The hP4Hα1 coding gene is linked into a PEE6.4 expression vector carrying an antibiotic selection marker to construct a recombinant plasmid PEE12.4-Puro-hP4Hα1; The hP4HB coding gene is linked into an expression vector PEE6.4 to construct a recombinant plasmid PEE6.4-hP4HB; The recombinant plasmid PEE12.4-Puro-hP4Hα1 and the recombinant plasmid PEE6.4-hP4HB are double digested with Not I and BamH I, and then linked to obtain the recombinant plasmid expressing hP4Hα1 and hP4HB.

2. A recombinant engineering cell expressing triple helical structure type III recombinant human collagen, which is constructed by the construction method of claim 1.

3. Use of the recombinant engineering cell of claim 2 in the preparation of triple helical structure type III recombinant human collagen.

4. A method for preparing a recombinant human collagen type III expressed by CHO cells having a triple helix structure, characterized by, The step of fermenting and purifying the triple helical structure type III recombinant human collagen by using the recombinant engineering cell of claim 2.

5. Triple helical structure type III recombinant human collagen, which is prepared by the preparation method of claim 4.

6. Use of the triple helical structure type III recombinant human collagen of claim 5 in the preparation of functional biomaterials, cosmetics or medical devices.

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