Bacterial recombinant collagen, preparation method and application

Abstract

The present invention discloses a bacterial recombinant collagen protein, a preparation method and an application thereof, and relates to the field of genetic engineering technology. The amino acid sequence of the bacterial recombinant collagen protein is shown in SEQ ID NO.1. The present invention can effectively promote cartilage repair, can be used as an alternative collagen-like biomaterial platform, provides a new method for cartilage repair, osteoarthritis and other diseases, and can also be used as an important exogenous material in cartilage repair scaffolds.

Description

Technical Field

[0001] The present invention relates to the field of gene engineering technology, and in particular to a bacterial recombinant collagen protein, a preparation method and an application thereof. Background Art

[0002] Natural collagen extracellular matrix, or ECM for short, components are widely used in tissue engineering applications due to their inherent biocompatibility and biofunctionality. In addition to numerous biomedical applications, collagen is also used in the cosmetic, pharmaceutical, and food industries. However, its use remains limited due to the many challenges posed by collagen. In order to change its material properties, collagen often needs to be modified by adding chemical groups, such as methacrylates, after purification to make it suitable for specific uses. In some cases, the reaction conditions required to modify collagen have been found to produce unwanted gels or partial denaturation of collagen, resulting in significant changes in the fiber structure and a subsequent loss of desired properties such as bioactivity and biodegradability. In addition, these natural ECM proteins are problematic from a source perspective. Currently, the main source of natural collagen comes from animal tissues and is mainly composed of the most abundant type I, which may expose patients to immunogenic responses and fail to promote cartilage repair. Summary of the invention

[0003] To solve the above problems, the present invention provides a bacterial recombinant collagen, a preparation method and an application thereof, which can effectively avoid antigen immune response and promote cartilage repair.

[0004] The present invention is achieved through the following technical solutions:

[0005] A bacterial recombinant collagen, the amino acid sequence of which is shown as SEQ ID NO.1.

[0006] A gene sequence encoding the bacterial recombinant collagen, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0007] An expression vector comprises the nucleotide sequence shown in SEQ ID NO.2, and the nucleotide sequence is shown in SEQ ID NO.3.

[0008] A host cell comprising the above expression vector.

[0009] Preferably, the host cell is Escherichia coli BL21.

[0010] A method for preparing bacterial recombinant collagen comprises culturing the host cell in a culture medium, inducing expression and then purifying the protein to obtain the bacterial recombinant collagen.

[0011] Preferably, the protein purification method includes salting out, ultrafiltration, affinity chromatography and gel filtration chromatography.

[0012] The bacterial recombinant collagen is used in the preparation of cartilage repair and osteoarthritis drug preparations.

[0013] The bacterial recombinant collagen is used in preparing exogenous materials for cartilage repair.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] 1. The bacterial recombinant collagen of the present invention has the same stability and safety as natural type I collagen, can form a triple helix structure in the absence of hydroxyproline, and has a moderate molecular weight of the translated protein, which is easy to prepare.

[0016] 2. The bacterial recombinant collagen of the present invention effectively avoids a series of antigen immune responses.

[0017] 3. The bacterial recombinant collagen prepared by the present invention is expressed by Escherichia coli engineered bacteria, has a high expression level, is lower in cost than natural collagen, and is suitable for large-scale production.

[0018] 4. The bacterial recombinant collagen prepared by the method of the present invention can effectively promote cartilage repair and can be used as an alternative collagen-like biomaterial platform, providing a new method for cartilage repair, osteoarthritis and other diseases. In addition, it can also be used as an important exogenous material in cartilage repair scaffolds.

[0019] 5. The bacterial recombinant collagen preparation method of the present invention is suitable for industrial large-scale production, and the prepared product has no animal-derived infection source, so it has higher biosafety. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 This is the SDS-PAGE protein electrophoresis diagram of the target bacterial recombinant collagen obtained in Example 1 of the present invention; wherein, from left to right, they are Markers; Lane 1: before induction; Lane 2: after induction; Lane 3: supernatant after centrifugation; Lane 4: precipitate after centrifugation;

[0022] Figure 2 The SDS-PAGE protein electrophoresis diagram of the protein supernatant under different volumes of ammonium sulfate and the SDS-PAGE protein electrophoresis diagram of the protein supernatant under different pH in Example 2 of the present invention;

[0023] Figure 2 A: SDS-PAGE protein electrophoresis of protein supernatant under different volumes of ammonium sulfate, from left to right are lanes 1 to 11: Marker, original sample, ammonium sulfate volume 10% to 90%; B: SDS-PAGE protein electrophoresis of protein supernatant under different pH, from left to right are lanes 1 to 12: Marker, original sample, Ph1 to 10;

[0024] Figure 3 This is a diagram of the target protein components collected after SDS-PAGE electrophoresis detection in Example 2 of the present invention;

[0025] Figure 4 AO / EB staining images of the collagen-free group, natural type I collagen group and bacterial recombinant collagen group of the present invention;

[0026] Figure 4 In the figure, A: AO / EB staining of the group without added collagen; B: AO / EB staining of the group with natural type I collagen; C: AO / EB staining of the group with bacterial recombinant collagen. DETAILED DESCRIPTION

[0027] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively below, and preferred embodiments of the present invention are provided. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0029] The inventive concept of the present invention is as follows:

[0030] Streptococcus pyogenes, a Gram-positive bacterium, produces a cell surface collagen-like protein called Scl2 that may help the pathogen adhere to animal cells. Sc12 was found to be acidic and highly charged. The collagen-like CL domain, composed of the repeating amino acid triplets Gly-Xaa-Yaa, is characteristic of collagen and is bracketed by the N-terminal assembly domain V and the C-terminal transmembrane domain. Notably, the CL domain is highly charged and stabilizes the triple helical structure in the absence of hydroxyproline by forming intermolecular salt bridges between the charged amino acids. Subdomains of the Sc12 CL domain have been expressed individually as homodimers and homotrimers fused to the N-terminal V domain to form a stable helical structure, the stability of which was found to depend on the amino acid composition, sequence length, and mutation position in the native sequence.

[0031] A sequence of natural bacterial collagen was repeated three times to form the recombinant bacterial collagen-like sequence in the present invention, denoted as eVB3m, which consists of three repeats of the charge-modified Bm subdomain, which is based on the Sc12 CL domain sequence fused to the natural N-terminal V domain and 6-His tag. A modified version with a C-terminal GFPGER integrin binding sequence motif was created using site-directed mutagenesis. The constructed collagen can specifically recognize integrins, thereby enhancing adhesion and promoting cartilage repair, and is comparable to natural collagen, and is easy to prepare and low cost.

[0032] The present invention provides a bacterial recombinant collagen, the amino acid sequence of which is shown in SEQ ID NO.1:

[0033] SEQ ID NO.1:

[0034] HHHHHHADEQEEKAKVRTELIQELAQGLGGIEKKNFPTLGDEDLDHTYMTKLLTYLQEREQAENSWRKRLLKGIQDHALDLVPRGSPGPKGEQGPQGLPGKDGEAGAQGPAGPMGPAGEQGEKGEPGTQGAKGDRGETGPKGPKGERGEAGPAGKDGERGPVG PAGPKGEQGPQGLPGKDGEAGAQGPAGPMGPAGEQGEKGEPGTQGAKGDRGETGPKGPKGERGEAGPAGKDGERGPVGPAGPKGEQGPQGLPGKDGEAGAQGPAGPMGPAGEQGEKGEPGTQGAKGDRGETGPKGPKGERGEAGPAGKDGERGPVGPAGFPGER

[0035] The present invention provides a bacterial recombinant collagen protein, which is screened for codons in host cell expression, and signal peptide cleavage sites and KpnI and BamHI restriction sites are added at both ends during the design process to facilitate later gene manipulation. After the above screening, its nucleotide sequence is shown in SEQ ID NO.2:

[0036] SEQ ID NO.2:

[0037] CACCACCATCACCACCACGCTGACGAACAAGAAGAGAAAGCGAAAGTTCGTACCGAACTGATCCAAGAACTGGCGCAGGGTTTGGGTGGTATCGAG AAGAAGAACTTTCCGACTCTGGGTGACGAAGACCTGGACCACACCTACATGACCAAACTGCTGACCTACCTGCAAGAACGTGAACAGGCGGAGAACTCTTGGCGTAAACGTCTGCTGAAAGGTATCCAAGACCACGCACTGGACCTGGTTCCGCGTGGTTCTCCGGGTCCGAAGGGTGAGCAAGGTCCGCAGGGTCTTCCGGGCAAAGATGGTGAAGCAGGTGCGCAGGGACCGGCAGGTCCAATGGGTCCGGCTGGTGAACAGGGCGAGAAAGGCGAACCGGGTACTCAGGGTGCTAAAGGTGATCGTGGTGAAACCGGTCCAAAGGGTCCGAAGGGAGAACGTGGTGAGGCAGGTCCGGCGGGTAAAGATGGCGAGCGTGGTCCAGTAGGTCCAGCTGGTCCGAAGGGCGAACAGGGTCCGCAAGGTCTGCCAGGCAAAGACGGTGAAGCGGGTGCTCAAGGTCCAGCCGGTCCAATGGGACCGGCGGGTGAACAGGGTGAGAAGGGTGAACCAGGTACTCAAGGTGCGAAAGGCGACCGTGGCGAAACCGGACCTAAAGGTCCAAAGGGTGAACGCGGTGAGGCTGGTCCGGCAGGTAAAGACGGCGAACGTGGACCAGTTGGTCCAGCTGGCCCTAAAGGCGAACAAGGTCCACAGGGTCTGCCGGGTAAAGACGGAGAAGCTGGTGCTCAGGGTCCAGCAGGTCCGATGGGACCAGCAGGTGAACAAGGTGAGAAAGGTGAACCGGGTACGCAGGGTGCAAAGGGTGACCGTGGTGAGACCGGTCCGAAAGGTCCGAAAGGTGAGCGTGGTGAAGCTGGTCCAGCGGGTAAAGACGGTGAACGTGGTCCGGTTGGTCCGGCTGGCTTTCCGGGTGAACGT

[0038] The present application provides an expression vector comprising the above-mentioned nucleotide sequence.

[0039] The vector may contain regulatory sequences, such as transcriptional and translational initiation and termination codons, which are specific for the type of host into which the vector is to be introduced, for example, bacteria, fungi, plants or animals, as appropriate and taking into account whether the vector is DNA or RNA based.

[0040] In a specific embodiment, the expression vector is pCold II, and its nucleotide sequence is shown in SEQ ID NO.3:

[0041] SEQ ID NO.3:

[0042]

[0043] The present invention provides a host cell comprising the above nucleotide sequence.

[0044] The host cell refers to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include transformants and transformed cells, which include primary transformed cells and progeny derived therefrom, regardless of the number of passages. Progeny may not be completely identical to parent cells in nucleic acid content, but may contain mutations.

[0045] In a specific embodiment, the host cell is selected from any one of Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, and Bacillus subtilis.

[0046] In a specific embodiment, the host cell is Escherichia coli BL21.

[0047] The present application provides a method for preparing any of the aforementioned bacterial recombinant collagen, which comprises the following steps:

[0048] The above-mentioned host cells are used for expression and then separated and purified to obtain the product.

[0049] The term "expressing a host cell" refers to culturing the host cell. The culture medium and culture conditions are well known to those skilled in the art.

[0050] In a specific embodiment, the host cell is Escherichia coli. After the constructed plasmid is obtained, the specific conditions for preparing BL21 competent cells are as follows:

[0051] (1) Activate the E. coli BL21 strain, pick a single BL21 colony from a fresh LB plate cultured overnight at 37°C, inoculate it into 3 mL LB liquid medium, and culture it at 37°C and 200 rpm overnight;

[0052] (2) Take 1 mL of overnight culture solution and inoculate it into 100 mL of liquid LB medium. Cultivate at 37°C and 200 rpm until OD 600 =0.3~0.5;

[0053] (3) Transfer the culture medium to a sterilized 50 mL centrifuge tube and place on ice for 10 min; centrifuge at 4°C and 6000 rpm for 5 min, discard the supernatant; resuspend the bacterial cells in 10 mL of pre-cooled sterile TSS;

[0054] (4) Place on ice for 10 min. Aliquot and store at -80°C until ready for use.

[0055] In a specific embodiment, the specific conversion conditions are as follows:

[0056] (1) Take a small amount of recombinant plasmid and an appropriate amount of BL21 competent cells and gently mix them, and place them on ice for 30 minutes;

[0057] (2) After the ice bath, heat shock at 42°C for 45 to 60 seconds, and immediately cool to room temperature in ice;

[0058] (3) Add an appropriate amount of LB liquid medium without antibiotics, mix gently, and culture at 37°C and 220 rpm for 1 h to allow the bacteria to fully recover;

[0059] (4) Spread the recovery solution evenly on an LB plate containing the corresponding antibiotics and culture it inverted at 37°C overnight.

[0060] In a specific embodiment, the specific induction expression conditions are as follows:

[0061] (1) Pick the positive transformants on the LB plate, inoculate them into LB medium, culture them overnight, and add resistance.

[0062] (2) Inoculate the activation solution into the expansion medium at a 1% inoculation rate and wait until OD 600 When the cells reached the logarithmic growth phase, IPTG was added to a final concentration of 0.5 mM and induced at 18°C ​​for 16 h, and resistance was added.

[0063] (3) Collect the fermentation liquid in a centrifuge tube, centrifuge at 8000 r / min for 10 min, remove the supernatant, resuspend in PBS, and use an ultrasonic crusher to break it. After breaking, centrifuge and take the supernatant, prepare the sample and perform SDS-PAGE protein electrophoresis.

[0064] The present application does not impose any limitation on the separation and purification method, which can be determined according to the method, for example, salting-out method, ultrafiltration method, affinity chromatography method and gel filtration chromatography method can be used.

[0065] The present invention also provides that the bacterial recombinant collagen, or the bacterial recombinant collagen encoded by the nucleic acid molecule, or the bacterial recombinant collagen expressed by the expression vector, or the bacterial recombinant collagen produced by the host cell has the ability to promote cartilage repair.

[0066] In order to better illustrate the present invention, the technical solution of the present invention will be further described below in conjunction with specific embodiments.

[0067] Example 1. Expression of bacterial recombinant collagen

[0068] (1) Chemically synthesize the bacterial recombinant collagen gene of the present invention, the nucleotide sequence of which is shown in SEQ ID NO.2. A sequence of natural bacterial collagen was repeated three times to form the recombinant bacterial collagen-like sequence of the present invention, represented as eVB3m, which consists of three repeats of the charge-modified Bm subdomain, which is based on the Sc12 CL domain sequence fused to the natural N-terminal V domain and 6-His tag. The bacterial recombinant collagen codon-optimized sequence constructed in the present invention, as shown in SEQ ID NO.2, was cloned into pCold II using KpnI and BamHI. A modified version with a C-terminal GFPGER integrin binding sequence motif was also created using site-directed mutagenesis to obtain the pCold II-eVB3m-GFPGER recombinant plasmid.

[0069] (2) Take a small amount of pCold II-eVB3m-GFPGER recombinant plasmid and an appropriate amount of BL21 competent cells and gently mix them, and place them in an ice bath for 30 minutes; after the ice bath, heat shock them at 42°C for 45s-60s, and immediately put them in ice to cool to room temperature; add an appropriate amount of LB liquid culture medium without antibiotics, gently mix them, and culture them at 37°C and 220r / min for 1h to fully revive the bacteria; evenly spread the recovery liquid on the LB plate containing the corresponding antibiotics, and culture them upside down at 37°C overnight. Pick the positive transformants on the LB plate, inoculate them in LB culture medium and culture them overnight, add resistance, and obtain the expression engineered bacteria pCold II-eVB3m-GFPGER-BL21.

[0070] (3) Inoculate the activation solution into the expansion medium at a 1% inoculation rate and wait until OD 600 When the cells reach the logarithmic growth phase, add IPTG at a final concentration of 0.5 mM and induce at 18°C ​​for 16 h, and add resistance. Collect the fermentation broth in a centrifuge tube, centrifuge at 8000 r / min for 10 min, remove the supernatant, resuspend in PBS, and use an ultrasonic crusher to break the supernatant. After breaking, centrifuge to take the supernatant, prepare the sample, and perform SDS-PAGE protein electrophoresis to obtain the target bacterial recombinant collagen, such as Figure 1 shown.

[0071] Example 2: Purification of bacterial recombinant collagen

[0072] (1) After the supernatant obtained by centrifugation in Example 1 is harvested, it is purified by ammonium sulfate precipitation and pH graded precipitation. pH graded precipitation: the solubility of protein is lowest at the isoelectric point and various proteins have different isoelectric points for separation, and the pH is set to 1-10; ammonium sulfate precipitation: the protein sample is added to a buffer containing ammonium sulfate at an ammonium sulfate content of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90%. Place in a 4°C refrigerator for 4 hours and centrifuge at 10000r / min for 10 minutes to separate the supernatant, reconstitute the precipitate with pure water, and perform SDS-PAGE protein electrophoresis on the protein supernatants under different volumes of ammonium sulfate and at different pH. Figure 2 shown.

[0073] (2) The crude pure solution obtained under appropriate conditions is further filtered through a 0.22 μm filter membrane. 2+ The pre-packed column of ions was purified using the AKTA system. AKTA operation process: first rinse with a pH 7.4 equilibrium buffer until the baseline is stable, then elute with an eluent, the flow rate is set to 5 mL / min, the UV detection wavelength is 215 nm, and finally collect the effluent target protein components after SDS-PAGE electrophoresis detection, such as Figure 3 shown.

[0074] The composition of the equilibration buffer was: 20 mM sodium phosphate + 20 mM imidazole + 500 mM NaCl.

[0075] The composition of the eluent: 20 mM sodium phosphate + 500 mM imidazole + 500 mM NaCl.

[0076] (3) Ultrafiltration desalting: Desalting is done by using a G25 desalting column, i.e., using 25 mL of G25 filler. The operation process is similar to the gel filtration chromatography step. 6.5 mL of sample is loaded each time, and about 8 mL is collected. Desalting is completed 10 minutes after loading.

[0077] (4) The protein was concentrated to 20% to 30% of the initial volume by ultrafiltration, and then pre-frozen in a -20°C refrigerator for 4 h. The protein was then transferred to a vacuum freeze dryer for freeze drying. The freeze-dried protein was collected after 72 h and stored in a 4°C refrigerator for later use.

[0078] Example 3: Experiment on promoting cell proliferation by bacterial recombinant collagen

[0079] The in vitro cell proliferation experimental model was used to evaluate the cell activity induced by bacterial recombinant collagen. Briefly, chondrocytes in logarithmic growth phase were seeded in 96-well tissue culture plates and cultured at 37°C in 5% CO. 2 Culture in a biochemical incubator for 1 day.

[0080] The in vitro cell proliferation experimental model was used to evaluate the cell activity induced by bacterial recombinant collagen. Briefly, chondrocytes in logarithmic growth phase were seeded in 96-well tissue culture plates and cultured at 37°C in 5% CO. 2 Cultured in a biochemical incubator for 1 day. The old culture medium was aspirated, and the natural type I collagen was dissolved in DMEM culture medium, filtered and sterilized and added to the above 96-well plate, with 100ul per well as the natural type I collagen group; the purified bacterial recombinant collagen was dissolved in DMEM culture medium, filtered and sterilized and added to the above 96-well plate, with 100ul per well as the experimental group, and the negative control group was 100ul of fresh complete culture medium without collagen addition, with five parallels per group. After incubation for 24 hours, the CCK8 method was used to verify cell proliferation, and the absorbance was measured at 450nm, and the cell proliferation rate was calculated respectively, and the culture medium without collagen addition, that is, the negative control group, was set as 100%. The results are shown in Table 1.

[0081] Table 1 Cell proliferation rate

[0082] Negative control group Natural type I collagen group Experimental Group Proliferation rate (%) 100％ 114％ 117％

[0083] At the same time, the negative control group, natural type I collagen group and experimental group were stained with AO / EB, and their growth after incubation in collagen culture medium was observed under a fluorescence microscope. The AO / EB staining images are shown in Figure 2. Figure 4 shown.

[0084] The results show that the bacterial recombinant collagen of the present invention is non-toxic and can significantly promote the proliferation of chondrocytes. The proliferation-promoting effect is similar to that of natural collagen and is significantly higher than that of the culture medium without additives. It has good cell compatibility and exhibits a good promoting effect, which is consistent with the results of light microscopy observation.

[0085] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0086] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A bacterial recombinant collagen, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

1.

2. A gene encoding the bacterial recombinant collagen as claimed in claim 1, characterized in that: The nucleotide sequence is shown in SEQ ID NO.

2.

3. An expression vector, characterized in that: It comprises the nucleotide sequence as claimed in claim 2, and the nucleotide sequence is shown as SEQ ID NO.

3.

4. A host cell, characterized in that Contains the expression vector as claimed in claim 3.

5. The host cell according to claim 4, characterized in that The host cell is Escherichia coli BL21.

6. A method for preparing bacterial recombinant collagen, characterized in that: The host cell according to claim 4 or claim 5 is cultured in a culture medium, and protein purification is performed after inducing expression to obtain bacterial recombinant collagen.

7. The method for preparing bacterial recombinant collagen according to claim 6, characterized in that: The protein purification method includes salting-out method, ultrafiltration method, affinity chromatography method and gel filtration chromatography method.

8. Use of the bacterial recombinant collagen as claimed in claim 1 in the preparation of cartilage repair pharmaceutical preparations.

9. Use of the bacterial recombinant collagen as claimed in claim 1 in preparing exogenous materials for cartilage repair.

Images