WO2022237224A1 - 一种重组人xvii型胶原蛋白、制备方法和应用 - Google Patents

一种重组人xvii型胶原蛋白、制备方法和应用 Download PDF

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WO2022237224A1
WO2022237224A1 PCT/CN2022/071968 CN2022071968W WO2022237224A1 WO 2022237224 A1 WO2022237224 A1 WO 2022237224A1 CN 2022071968 W CN2022071968 W CN 2022071968W WO 2022237224 A1 WO2022237224 A1 WO 2022237224A1
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collagen
seq
amino acid
tag
cell
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PCT/CN2022/071968
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French (fr)
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李佳佳
王丽萍
钱松
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江苏创健医疗科技有限公司
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Priority to JP2022548165A priority Critical patent/JP7411858B2/ja
Priority to KR1020227028808A priority patent/KR102559311B1/ko
Priority to EP22744363.7A priority patent/EP4119573A4/en
Priority to US17/908,258 priority patent/US11787852B2/en
Publication of WO2022237224A1 publication Critical patent/WO2022237224A1/zh

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    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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Definitions

  • the invention relates to a recombinant human type XVII collagen, a preparation method and application, and belongs to the technical field of genetic engineering.
  • collagen As an important natural biological protein, collagen can be widely used in many fields such as chemical industry, medicine, food, cosmetics, etc. It is especially suitable for the preparation of various biological devices. It is the most ideal source of biological materials and has broad application prospects.
  • Collagen sold in the market is mainly the collagen extract obtained by treating animal tissues with acid, alkali and enzymatic hydrolysis. During the processing of such extracts, the natural structure of collagen is basically destroyed, the degradation is severe, and its biological activity is lost; the extracted collagen peptides have different lengths, uneven properties, unstable quality, and the risk of virus infection; at the same time, animals The amino acid sequence of human-derived collagen differs greatly, which can lead to immune rejection and allergic symptoms.
  • Human type XVII collagen is a transmembrane non-fibroblast collagen, which is a homogeneous trimer composed of three identical ⁇ 1(XVII) chains, and is divided into three types of intracellular, transmembrane and extracellular domains.
  • Human type XVII collagen is a component of hemidesmosome in cells, which plays an important role in the interaction between epithelial cells and basement membranes, and can regulate the adhesion, separation, development and differentiation of epithelial cells, and the differentiation and regeneration of keratinocytes have an important role.
  • the content of human type XVII collagen is very small in the human body, and the content of type XVII collagen in animals is also extremely rare, so it is very difficult to extract and cannot be mass-produced.
  • prokaryotic (E. coli) expression system prokaryotic (E. coli) expression system
  • Pichia pastoris expression system mammalian cell expression system
  • insect cell (baculovirus) expression system insect cell (baculovirus) expression system.
  • mammalian cells have high cost and low yield, and are mostly used for the expression and production of high-value pharmaceutical proteins.
  • Human type XVII collagen expressed by mammalian cells whether it is expressed by transient transfection or stably transfected cell lines, the protein expression level is extremely low, and the medium used is expensive, which can only meet the trace use in scientific research.
  • Insect cell (baculovirus) expression system is not only high in cost and low in yield, but its post-translation is very different from that of human cells. These two methods are generally not used in the mass production and application of collagen.
  • Prokaryotic expression (Escherichia coli expression system) of collagen has no post-translational modification of the protein, and prokaryotic expression with large-scale amplification prospects can only be intracellular expression, which needs to be lysed by bacteria, and there will be a large amount of impurity proteins mixed with the target protein , has extremely high requirements on the purification process, and the prokaryotic expression system naturally contains (bacterial cell wall components) endotoxin and peptidoglycan, which require complex purification processes to remove.
  • Human type XVII collagen belongs to transmembrane collagen, which has intracellular region, transmembrane region and extracellular region.
  • transmembrane proteins when transmembrane proteins are expressed in eukaryotic cells, most of the time they are not secreted outside the cell, but are fixed on the cell membrane, and the amino acid sequence of human type XVII collagen is very long (1497 amino acids), and the protein molecular weight Larger (180kDa), theoretically it is difficult to effectively secrete extracellularly and is easy to degrade. To successfully express it, it is necessary to select related sequences.
  • the purpose of the present invention is to overcome some technical problems in the prior art, mainly to overcome the optimal selection of recombinant human type XVII collagen sequence (non-uniqueness) and whether it can be secreted and expressed efficiently in Pichia pastoris, the existing recombinant Human type XVII collagen has only cell adhesion activity, and the existing recombinant human type XVII collagen cannot be produced on a large scale.
  • the present invention provides a collagen, a polynucleotide encoding the collagen, a recombinant vector containing the polynucleotide encoding the collagen, engineering bacteria, a method for preparing the collagen, and a composition comprising the collagen , the application of the collagen. It has been confirmed in the present invention that the collagen can not only realize the high-efficiency secretion and soluble expression of human type XVII collagen in eukaryotic host cells such as Pichia pastoris, but also has better cell adhesion than commercial natural human collagen activity, promoting cell migration, excellent tissue regeneration and biological activity promoting hair follicle repair and regeneration, and can be applied industrially.
  • the invention provides a collagen, and the collagen has the biological activities of cell adhesion activity, cell migration promotion activity, tissue regeneration promotion and hair follicle repair regeneration promotion.
  • the collagen protein consists of or comprises the amino acid sequence shown in (A) n , wherein: A is the amino acid sequence shown in SEQ ID NO.2 or based on SEQ ID NO.2 amino acid sequence modified to a certain extent by amino acid substitutions, insertions, substitutions, additions, deletions, etc., or an amino acid sequence that is more than 80% homologous to the amino acid sequence of SEQ ID NO.2; where n is greater than or equal to 1 Integer; wherein each A is the same or different and repeats in tandem with A as the basic unit, and each of the same or different A is directly connected by a peptide bond.
  • the collagen consists of or comprises the amino acid sequence shown in (A) n : wherein A is the amino acid shown in SEQ ID NO.2 Sequence; n is an integer greater than or equal to 1; wherein each A is the same and repeats in tandem with A as the basic unit, and each A is directly connected by a peptide bond.
  • the collagen is composed of the amino acid sequence shown in (A) n , when n is 1, the amino acid sequence of the collagen is shown in SEQ ID NO.2, this It is recorded as 170801 in the invention; when n is 2, the amino acid sequence of the collagen is shown in SEQ ID NO.3, and it is recorded as 170802 in the present invention.
  • the collagen of the present invention consists of or comprises the amino acid sequence shown in (A) n , wherein A is based on SEQ ID NO.2 An amino acid sequence modified to a certain extent by amino acid insertion, substitution, addition, deletion, etc., or an amino acid sequence with greater than 80% homology to the amino acid sequence of SEQ ID NO.2; where n is an integer greater than or equal to 1; where each The A's are the same or different and repeat in tandem with the A as the basic unit, and each of the same or different A's is directly connected by a peptide bond.
  • the present invention also provides a polynucleotide encoding the above-mentioned collagen, specifically a polynucleotide sequence encoding the collagen represented by (A) n ; preferably, when Pichia pastoris is used as a host cell, the polynucleotide comprises The nucleotide sequence shown in SEQ ID NO.6 or SEQ ID NO.7 or consists of the nucleotide sequence shown in SEQ ID NO.6 or SEQ ID NO.7.
  • said A is SEQ ID NO.2, and the polynucleotide when n is 1 is the nucleotide sequence shown by SEQ ID NO.6; According to one embodiment of the present invention, the Said A is SEQ ID NO.2, when n is 2, the polynucleotide encoding the above (A)n is the nucleotide sequence shown in SEQ ID NO.7.
  • the collagen of the present invention is a combination sequence selected after optimization and screening from multiple helical region sequences such as the 15th helical region, the carboxyl terminal region, and the middle region of human type XVII collagen, and the corresponding part of the amino acid sequence of human type XVII collagen 100% identical, not only can realize the high-efficiency secretion and soluble expression of human type XVII collagen in eukaryotic host cells such as Pichia pastoris, but also have better cell adhesion activity and promote cell migration than commercial natural human collagen activity and the biological activity of promoting tissue regeneration and hair follicle repair and regeneration; such a combined sequence enables the integration of the functions of the sequences in each region, not expressed separately but as a whole, avoiding the sequence unity of recombinant collagen .
  • the present invention also provides a recombinant expression vector containing the polynucleotide encoding the above collagen.
  • the polynucleotide encoding the collagen represented by (A) n is modified, and the amino acid sequence of the collagen is respectively added with a coding
  • the DNA sequence of the Strep-Tag II tag and the DNA sequence encoding the 6 ⁇ His Tag tag are added to the carboxy-terminus to make it contain a bispecific affinity purification tag, which can be purified by affinity chromatography, and it is also convenient to use the two tag sequences as Basic immunological antibody detection.
  • the marked sequence was ligated into the expression vector pPIC9K to construct a recombinant expression vector (represented as pPIC9K-170801 and pPIC9K-170802 in the examples).
  • the present invention also provides an engineering bacterium constructed by the above-mentioned recombinant expression vector, and the host cell of the engineering bacterium is preferably Pichia pastoris.
  • the preservation date of the engineering bacteria is September 9, 2020, the preservation numbers are CGMCC NO.20626 and CGMCC NO.20627, and the classification is named Pichia pastoris, and the preservation unit is the China Microbiological Culture Collection Management Committee Ordinary Microbiology Center is located at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.
  • the host cells can be eukaryotic cells, such as fungi, yeast, plant cells, etc.; prokaryotic cells, such as Enterobacteriaceae bacteria, such as Escherichia coli; or animal cells, such as mammalian cells CHO series cells, HEK293 Serial cells or insect cells. It should be understood that those skilled in the art can express the collagen with the same amino acid sequence by replacing the above-mentioned Pichia pastoris with other expression hosts.
  • the present invention also provides the preparation method of above-mentioned collagen protein, comprises the following steps:
  • the engineering bacterium with high expression level screened is Pichia pastoris, and the preservation number is CGMCC No.20626 or CGMCC No.20627.
  • 50L-500L fermentation tanks are used for joint fed-batch fermentation, and the fermentation supernatant is purified to obtain high-purity collagen.
  • the present invention uses Sac I to linearize the recombinant expression vector, electroporate into Pichia pastoris competent cells, construct engineering bacteria, and transfer the engineering bacteria to the MD plate for primary screening, and then pass through the process containing different concentrations G418 was screened on the YPD plate, and colonies were picked and inserted into BMGY medium, and then induced to express with BMMY medium; multiple strains were picked, and engineering bacteria with high expression levels were selected for subsequent experiments.
  • the expressed protein is initially identified by SDS-PAGE electrophoresis and Western Blot, and its ability to effectively secrete and express is judged.
  • Type XVII collagen is mainly concentrated in the cell and cannot be effectively secreted outside the cell; the target band obtained by electrophoresis is detected by Nano-HPLC-MS/MS mass spectrometry, and it is proved that the peptide is derived from the optimally selected human type XVII collagen sequence Region, the recombinant human type XVII collagen 170801 and 170802 of the present invention were successfully expressed.
  • the collagen production (UV quantification) of the present invention can reach 12g/L, 11g/L.
  • MALDI-TOF/TOF is used to measure the molecular weight.
  • the molecular weight of 170801 collagen is 23811.6797Da (theoretical value is 23800Da), and the molecular weight of 170802 collagen is 45689.5781Da (theoretical value is 45400Da).
  • There are systematic errors in post-translational modification and molecular weight experimental determination and the actual expressed collagen molecular weight is consistent with the theoretical value.
  • the wavenumbers of amide A, amide B, amide I, amide II, and amide III all conform to the structural characteristics of recombinant collagen.
  • the purified collagen freeze-dried sponge scanned by electron microscope has a lamellar structure and has the potential to be used in the field of biomedical materials.
  • the biological activity of the obtained collagen is detected, and its cell adhesion activity and cell migration promotion activity are verified by in vitro cell experiments.
  • Animal experiments on the biological activity of the obtained collagen, rabbit ear scar model experiments have verified that it can effectively promote the healing and tissue regeneration of rabbit ear scars, especially the extremely excellent activity of promoting hair follicle repair and regeneration.
  • the present invention also provides the use of the above-mentioned collagen in promoting tissue regeneration and hair follicle repair.
  • the present invention also provides a composition, which comprises the above collagen or the collagen prepared by the above method.
  • the present invention also provides a product, which contains the above-mentioned collagen or the collagen prepared by the above-mentioned method or the above-mentioned composition.
  • the products include, but are not limited to, drugs, pharmaceutical compositions, medical devices, biological materials, tissue engineering products, cosmetics or health care products, and the like.
  • the present invention also provides the use of the collagen, nucleotide, recombinant expression vector, engineering bacteria, composition, etc. in the preparation of finished products, including but not limited to drugs, pharmaceutical compositions, medical equipment, biological materials, tissue engineering Use in products, cosmetics, and health care products.
  • the present invention also provides the use of the collagen, nucleotide, recombinant expression vector, engineering bacteria, or composition in the preparation of products for promoting scar healing, tissue repair or hair follicle repair.
  • the product is an external preparation, preferably an external application preparation, preferably a gel.
  • the present invention selects the expression system of Pichia pastoris.
  • Pichia pastoris is a eukaryotic microorganism with complete organelles of eukaryotic organisms, and can carry out certain post-translational modifications (especially glycosylation modification) on translated proteins, which is powerful Support the realization of protein biological functions. With its establishment of expression system, it has the advantages of high-density fermentation production, extremely low cultivation cost, short cycle, high expression and other large-scale industrial production advantages of microbial expression system; it can be secreted extracellularly and can completely lyse bacteria The impurity protein brought; the cell wall components do not contain endotoxin and peptidoglycan.
  • the sequence selected for recombinant human type XVII collagen in the present invention is a combination of optimized sequences through screening, and its amino acid sequence is 100% homologous to the corresponding part of the amino acid sequence of natural collagen, has no immunogenicity, and can be used widely. Used in medicine, medical equipment, biomaterials, tissue engineering, cosmetics and other fields.
  • the recombinant human type XVII collagen in the present invention has been verified by experiments, and has the same or better cell adhesion activity, cell migration activity, tissue repair and promotion of hair follicle repair and regeneration as commercialized natural human collagen. Learning activity can achieve the purpose of real product application.
  • the recombinant human type XVII collagen in the present invention can be efficiently secreted and expressed in the Pichia pastoris expression system, and it is easy to purify to obtain high-purity collagen; the DNA sequence of the gene encoding the amino acid sequence has been optimally designed, so that the constructed engineering strain The protein expression is very high, and a high yield can be obtained during high-density fermentation expression; Pichia pastoris is easy to perform high-density fermentation expression, and the strains screened by the present invention have been able to achieve 500L pilot-scale high-density high-expression fermentation , already has the conditions for industrialized mass production.
  • the host cell Pichia pastoris in the present invention has certain post-translational modifications on the expressed foreign protein, such as glycosylation and the like.
  • Fig. 1 is a map of the pPIC9K-170801 vector constructed in the present invention.
  • Fig. 2 is a vector map of pPIC9K-170802 constructed by the present invention.
  • Fig. 3 is a map of the pPIC9K-col17a1 vector constructed in the present invention.
  • Figure 4 is the SDS-PAGE result of the expression of recombinant full-length human type XVII collagen ⁇ 1 chain, the apparent molecular weight of which is about 180kDa.
  • Figure 5 shows the Western Blot detection results of recombinant full-length human type XVII collagen ⁇ 1 chain using anti-His tag antibody.
  • Fig. 6 is the result of SDS-PAGE of the supernatants expressing the engineered bacteria of 170801 and 170802 collagen after 24 hours of induction.
  • Figure 7 shows the results of Western Blot detection of the supernatants expressed by engineered bacteria of 170801 and 170802 collagen after 24 hours of induction using anti-His tag antibodies.
  • Figure 8 shows the results of Western Blot detection of the supernatants expressed by engineered bacteria of 170801 and 170802 collagen after 24 hours of induction using anti-Strep-Tag II tag antibodies.
  • Fig. 9 is the comparison result of the mass spectrometry analysis of the SDS-PAGE band of 170801 collagen and the sequence of natural human type XVII collagen.
  • Fig. 10 is the comparison result of the mass spectrometry analysis of the SDS-PAGE band of 170802 collagen and the sequence of natural human type XVII collagen.
  • Figure 11 is the growth curve and collagen expression curve of 171801 collagen engineered bacteria in a 500L fermenter.
  • Figure 12 is the growth curve and collagen expression curve of 171802 collagen engineered bacteria in a 500L fermenter.
  • Figure 13 is the HPLC profile of purified 170801 collagen.
  • Figure 14 is the HPLC profile of purified 170802 collagen.
  • Figure 15 shows the MALDI-TOF MS detection results of the molecular weight of 170801 collagen.
  • Figure 16 is the MALDI-TOF MS detection result of the molecular weight of 170802 collagen.
  • Figure 17 is the infrared spectrum of purified 170801 and 170802 collagen.
  • Figure 18 is the SEM images (200 ⁇ ) of 170801 (left), 170802 (right) collagen freeze-dried sponge samples.
  • Figure 19 is the test results of cell adhesion activity of 170801 and 170802 collagens.
  • Figure 20 is the actual comparison result of the cell migration state of 170801, 170802 collagen and natural human collagen.
  • Figure 21 shows the cell migration rate of 170801, 170802 collagen and natural human collagen after 24h and 48h of cell migration.
  • Fig. 22 is the general photo and scar section of the 6-week intervention of 170801 and 170802 collagen on rabbit ear scars.
  • the specific sequence of human type XVII collagen refers to: Uniprot Q9UMD9-1 sequence (https://www.uniprot.org/uniprot/Q9UMD9) and NCBI reference sequence Q9UMD9. 3 (https://www.ncbi.nlm.nih.gov/protein/Q9UMD9.3), the two sequences are identical, as shown in SEQ ID NO.1.
  • the bold underlined part of the above sequence SEQ ID NO.1 is the sequence selected by the present invention, with a total of 233 amino acids.
  • the sequence selected in the present invention is a combined sequence selected after optimization and screening from multiple helical region sequences such as the 15th helical region, the carboxyl terminal region, and the middle region of human type XVII collagen, which can not only realize the Eukaryotic yeast, a eukaryotic host cell, is highly secreted and soluble expressed, and it also has better cell adhesion activity, cell migration activity, excellent tissue regeneration and biological activity of promoting hair follicle repair and regeneration than commercial natural human collagen Such a combination enables the functions of the sequences in each region to be integrated, and is not expressed separately but as a whole, which avoids the sequence unity of the recombinant collagen and is highly innovative. It is named in the present invention. It is 170801, which is the preferred sequence of human type XVII collagen, and the specific sequence is shown in SEQ ID NO.2.
  • the collagen in the present invention also includes the amino acid sequence obtained by taking 170801 as the basic unit and repeating n times in tandem, wherein n is an integer greater than or equal to 1.
  • 170801 When 170801 is used as the basic unit, when n is 2, 170801 is repeated twice in tandem to obtain an amino acid sequence with a total of 466 amino acids, which is named as 170802, and its amino acid sequence is shown in SEQ ID NO.3.
  • the DNA sequence encoding 170802 is shown in SEQ ID NO.7.
  • the function of the protein requires the orderly arrangement of its amino acid residues to be realized. All the innovative biological activities of the recombinant human type XVII collagen in the present invention are based on the amino acid sequence selected by optimal screening, limited sequence modification or certain Proportional homology (greater than 80%) is still possible to achieve the same or similar biological activity.
  • the collagen also includes amino acid sequences modified to a certain extent on the basis of SEQ ID NO.2, such as amino acid insertion, substitution, addition, deletion, etc.
  • the homology in the present invention refers to the overlap rate of sequences, which can be the direct quantitative relationship between the two sequences, such as the percentage of partial identity and similarity; it also includes the nucleotide or amino acid residues at the same position of the two sequences The exact same sequence ratio; it can also be a different sequence formed by divergent evolution from a common ancestor. It can be obtained by aligning sequence information with conventional bioinformatics methods.
  • the amino acid insertion refers to the insertion of amino acid residues at an appropriate position in an amino acid sequence, such as SEQ ID NO.2 or SEQ ID NO.3.
  • the rooms are not adjacent to each other.
  • the protein has biological activities similar to those of 170801 and 170802 collagens, including cell adhesion activity, cell migration promotion activity, tissue regeneration promotion activity and hair follicle repair regeneration activity.
  • amino acid substitution refers to one or several amino acid residues (including several consecutive amino acid residues or non-contiguous several amino acid residues) at a certain position in the amino acid sequence, such as SEQ ID NO.2 or SEQ ID NO.3 sequence residues) are replaced by other amino acid residues, as long as the protein has similar biological activities to 170801 and 170802 collagens, including cell adhesion activity, cell migration promotion activity, tissue regeneration promotion activity and hair follicle repair regeneration activity.
  • the amino acid addition refers to adding amino acids to the amino acid sequence, such as the C-terminal or N-terminal of the sequence of SEQ ID NO.2 or SEQ ID NO.3, as long as the protein has similar biological activity to 170801 and 170802 collagen, Including cell adhesion activity, promoting cell migration activity, promoting tissue regeneration and promoting hair follicle repair and regeneration activity.
  • the amino acid deletion means that 1, 2 or 3 or more than 3 amino acids can be deleted from the amino acid sequence, such as SEQ ID NO.2 or SEQ ID NO.3, as long as the protein has the same Similar biological activities include cell adhesion activity, cell migration activity, tissue regeneration promotion and hair follicle repair regeneration activity.
  • the amino acids described in the present invention are preferably L-type amino acids; at the same time, one or more (such as 2-5, 2-4 or 2-3) amino acids can also be artificially modified with D-type amino acids in conformation amino acids, rare amino acids that exist in nature, etc., to improve the bioavailability, stability and/or antiviral activity of the polypeptide.
  • D-type amino acids refer to amino acids corresponding to L-type amino acids that make up proteins
  • artificially modified amino acids refer to common L-type amino acids that make up proteins that have been modified by methylation, phosphorylation, etc.
  • rare amino acids that exist in nature include those that make up proteins.
  • the DNA sequences of 170801 and 170802 are modified by adding a DNA sequence encoding a Strep-Tag II tag to the amino terminus and a DNA sequence encoding a 6 ⁇ His Tag tag at the carboxyl terminus of the two, so that they contain bispecific Affinity purification tag, which can be purified by affinity chromatography and also facilitates immunological antibody detection based on two tag sequences.
  • the codon bias of DNA sequences and related optimization parameters in the process of transcription and translation are calculated, spliced, recombined and optimized. The synthesis is more suitable for Pichia pastoris highly expressed DNA sequences.
  • the two ends of 170801 are respectively modified, and after the DNA sequence encoding the Strep-Tag II tag is added to the amino terminus, and the DNA sequence encoding the 6 ⁇ His Tag tag is added to the carboxyl terminus, the final expression of 170801 is a tag-containing protein.
  • the optimized amino acid sequence of 170801 is shown in SEQ ID NO.4. Modified at both ends of 170801 respectively, adding DNA sequence encoding Strep-Tag II tag to the amino-terminus, DNA sequence encoding 6 ⁇ His Tag tag, stop codon (TAA) and DNA sequence of Not I restriction site to the carboxyl-terminus As shown in SEQ ID NO.8.
  • the final expression of 170802 is a tag-containing protein, a total of 482 amino acids, the optimized amino acid sequence of 170802 is shown in SEQ ID NO.5.
  • modify and add the DNA sequence encoding the Strep-Tag II tag at the amino-terminus add the DNA sequence encoding the 6 ⁇ His Tag tag, the stop codon (TAA) and the DNA sequence of the Not I restriction site at the carboxy-terminus as shown in SEQ ID NO.9.
  • the DNA sequence encoding the amino acid sequence of the full-length human type XVII collagen i.e. human type XVII collagen ⁇ 1 chain
  • the DNA sequence of the enzyme cleavage site of EcoR I added to the amino terminal, the Strep-Tag II tag, the carboxyl terminal Add the DNA sequence encoding the 6 ⁇ His Tag tag and the stop codon (TAA) and the restriction site of Not I) as shown in SEQ ID NO.10.
  • the optimized sequences SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10 were synthesized by Nanjing GenScript Biotechnology Co., Ltd., and the three gene fragments were denoted as pUC57-170801, pUC57- 170802, pUC57-col17a1.
  • the synthesized gene fragments SEQ ID NO.8 and SEQ ID NO.9 were double-digested with SnaB I and Not I, and the target gene fragments obtained after digestion were ligated into the same double-digested pPIC9K empty vector (purchased from Thermo Fisher Scientific Corporation).
  • the synthesized gene fragment SEQ ID NO.10 was double digested with EcoR I and Not I, and the target fragment obtained after digestion was ligated into the pPIC9K empty vector after the same double digestion, as a comparison.
  • Step a Amplify the target gene and expression vector: extract pUC57-170801, pUC57-170802, pUC57-col17a1, and pPIC9K empty vector plasmids with a plasmid mini-extraction kit (purchased from Sangon Bioengineering (Shanghai) Co., Ltd.), The specific operation was carried out according to the instructions of the kit.
  • a plasmid mini-extraction kit purchased from Sangon Bioengineering (Shanghai) Co., Ltd.
  • Step b Digest the target gene fragment obtained in step a and the pPIC9K vector plasmid at 37°C for 30 min.
  • the enzyme digestion reaction system is as follows (QuickCut TM restriction enzymes used were purchased from Dalian TaKaRa Company, and the enzyme digestion process was carried out according to its instructions. ):
  • the obtained target gene fragment and vector fragment were purified using a PCR product purification kit (purchased from Dalian TaKaRa Company, and the specific operation was performed according to the kit instructions).
  • the target gene fragment and vector pPIC9K linearized fragment after double enzyme digestion were recovered, ligated with Solution I ligation reagent (the ligation kit was purchased from Dalian TaKaRa Company) (ligated at 16°C for 30 min), and the target gene fragment was accurately inserted into the pPIC9K containing secretory
  • the secretion vector of the signal ⁇ -factor is in the reading frame, and the ligation reaction system is as follows (perform according to the ligation kit instructions):
  • the ligation product was transformed into competent Escherichia coli DH5 ⁇ (purchased from Sangon Bioengineering (Shanghai) Co., Ltd.), positive clones were screened on the LB resistance plate containing Amp, colony PCR verification was performed, and the recombinant plasmid was extracted for sequencing identification (submitted by Completed by Sangon Bioengineering (Shanghai) Co., Ltd.), the sequencing verification proved that the recombinant expression vectors pPIC9K-170801, pPIC9K-170802, and pPIC9K-col17a1 were successfully constructed in this example.
  • the relevant plasmid maps are shown in Figure 1, Figure 2 and Figure 2. 3 shown.
  • the Pichia transformant can grow on the plate containing high concentration of G418, it means that the transformant contains multiple copies of the target gene, that is, multiple recombination fragments have entered the yeast and integrated into the yeast chromosome through homologous recombination. After this step, the obtained high-copy, high-efficiency recombinant yeast engineered strains are screened.
  • the three engineering bacteria samples containing pPIC9K-col17a1, pPIC9K-170801, and pPIC9K-170802 were sent to the General Microbiology Center of China Microbiological Culture Collection Management Committee.
  • the strain preservation numbers are: CGMCC No.18659, CGMCC No.20626, CGMCC No. 20627. Address: No. 3, Courtyard No. 1, Beichen West Road, Chaoyang District, Beijing; date of preservation: CGMCC No.18659 is October 10, 2019, CGMCC No.20626, and CGMCC No.20627 are September 9, 2020.
  • Classification name Pichia pastoris.
  • the bacterial liquid samples were taken separately, the sampling volume was 1mL, placed in a 1.5mL EP tube, centrifuged at the maximum speed for 2-3min, and the expression supernatant and bacteria were collected. body.
  • the bacteria were lysed by the glass strain (purchased from Sangon Biotech (Shanghai) Co., Ltd.), centrifuged at 12000g for 5min at 4°C in lysis buffer (1mM PMSF, 10mM Tris, pH7.4), and the intracellular lysed supernatant was taken , and then add 8M urea to shake to dissolve the precipitate, and analyze the expression level of the target protein and the optimal harvesting time of the bacterial solution.
  • the samples to be tested were stored at -80°C for future use.
  • the expected target band (the apparent molecular weight is about 180 kDa) was not visible in the expression supernatant.
  • the experimental results show that: as a type of transmembrane collagen, it should be fixed on the membrane system in the cell, and can only be detached and dissolved in the cell after degradation, and the degraded target protein detected in the expression supernatant is extremely It may be released into the expression supernatant after lysing dead cells during long-term culture, but regardless of the possibility, the full-length recombinant human type XVII collagen cannot be effectively secreted outside the Pichia pastoris.
  • Figure 6 is the SDS-PAGE results of the supernatants expressed by the engineered bacteria of 170801 and 170802 collagen after 24 hours of induction.
  • the apparent molecular weight of 170801 is about 30kDa
  • the apparent molecular weight of 170802 is about 52kDa.
  • Figure 7 is the Western Blot result of the supernatant expressed by engineering bacteria of 170801 and 170802 collagen after 24 hours of induction (Anti-His, the antibody was purchased from Sangon Bioengineering (Shanghai) Co., Ltd., mouse anti-His monoclonal antibody, catalog number D199987 ),
  • Fig. 8 is the Western Blot result (Anti-Strep-Tag II, antibody purchased from Nanjing GenScript Biotechnology Co., Ltd., rabbit anti-Strep-Tag polyclonal antibody, Cat. No. A00626).
  • the engineered bacteria containing pPIC9K-170801 and pPIC9K-170802 respectively were fermented in a 50L-500L linkage pilot test to obtain a fermentation broth containing recombinant human type XVII collagen to achieve large-scale expression of recombinant human type XVII collagen 170801 and 170802 Production.
  • Seed medium YPG (formulation: yeast powder 10g/L, yeast peptone FP102 20g/L, anhydrous glycerin 10g/L); fermentation medium (formulation: NH 4 H 2 PO 4 190.4g/L, KH 2 PO 4 10.06g/L, CaSO 4 2H 2 O 1.18g/L, K 2 SO 4 18.2g/L, MgSO 4 7H 2 O 14.9g/L, glycerol 40g/L); feed medium (50% W/V glycerol, add 12mL PTM 1 trace elements per liter); induction medium (100% methanol, add 12mL PTM 1 trace elements per liter); wherein PTM 1 is sterilized by filtration with a 0.22 ⁇ m filter membrane, and stored at 4°C. After the fermentation medium was sterilized by high temperature, PTM 1 was added after the temperature dropped to room temperature, and the pH value was adjusted to 5.0 with ammonia water.
  • the batch culture conditions and induced expression conditions of the constructed engineering strains are:
  • a fed-batch culture method was adopted, and the culture temperature was 30°C.
  • the engineered bacterium of construction is connected in the shaking flask that seed medium YPG is housed, and the medicine bottle capacity is 2L, and 220rpm, 30 °C cultivate 24h; Inoculate in the seed fermenter that contains fermentation medium with 10% inoculum After inoculation, the OD600 is 5), the capacity of the fermenter is 50L, expand the cultivation, adjust the stirring speed to 200r/min-600r/min, the air flux is 2VVM, DO ⁇ 30%, cultivate until the carbon source is exhausted, transplant Start the fermentation culture in the fermenter containing the fermentation medium, and the capacity of the fermenter is 500L.
  • the fermentation culture conditions are: stirring speed 150r/min-500r/min, tank pressure 0-0.05MPa, air flux 1VVM, DO ⁇ 30%, dissolved oxygen rebound flow plus feeding medium, flow acceleration rate maintains DO ⁇ 30%, until the OD 600 grows to 190-210, stop feeding. After the dissolved oxygen rebounds to DO ⁇ 70%, feed the induction medium to induce expression, and adjust the rotation speed, ventilation, tank pressure and flow speed to make DO ⁇ 30%. After 40-72 hours of induction, the protein concentration measured by UV does not increase significantly or decreases, and then it can be put into the tank.
  • Buffer A 20mM KH 2 PO 4 , pH 4.0;
  • Buffer B 20 mM KH 2 PO 4 , 1 M NaCl, pH 4.0.
  • the obtained fermentation broth of the engineering bacteria was collected, and the bacterial body and the fermentation supernatant were separated using a ceramic membrane separation system (Jiangsu Jiuwu High-tech Co., Ltd., JWCMF-9).
  • Equilibrate the cation exchange medium with buffer A (the chromatographic filler is UniGel-80sp produced by Suzhou Nawei Technology, loaded in the automatic chromatography system produced by Jiangsu Hanbang Technology) until the A215 absorbance value and conductivity value remain unchanged, set up
  • the sample flow rate is 100us/cm
  • the sample volume is 20L/time
  • the ultraviolet A215 absorbance value is detected. When it rises, the sample is started.
  • the molecular weight of 170801 collagen detected by MALDI-TOF MS (charge number + 1) is 23811.6797Da (theoretical value is 23800Da), and the molecular weight of 170802 collagen is 45689.5781Da (theoretical value is 45400Da), due to the existence of post-translational modification after expression in Pichia pastoris and systematic errors in molecular weight experimental determination, the molecular weight of the actually expressed collagen is consistent with the theoretical value.
  • the characteristic absorption peaks of collagen groups can be detected by infrared spectroscopic analysis.
  • trace amounts of 170801 and 170802 collagen were purified and freeze-dried samples were ground into powders with KBr and pressed into tablets. At room temperature, they were scanned within the range of 4000-400cm Thermo Scientific, Nicolet TM iS TM 10FT-IR spectrometer), method and result analysis refer to (Jeong, H., J.Venkatesan and S.Kim, Isolation and characterization of collagen from marine fish (Thunnus obesus). Biotechnology and Bioprocess Engineering, 2013.18(6):p.1185-1191.)
  • NIH/3T3 cells were cultured normally (purchased from the Cell Bank of the Chinese Academy of Sciences, Cat. No. GNM6, and the cultivation and passage methods were performed according to the instructions of the cells).
  • the absorbance at OD492nm can correspond to the cell adhesion activity of the collagen sample: the higher the absorbance value, the more cells the protein adheres to, and the higher the adhesion activity, the more collagen can help cells adhere to the wall or adhere in a short time. Attached to the extracellular matrix, it is more conducive to building a better extracellular environment. Results As shown in Figure 19, the recombinant human type XVII collagen 170801 and 170802 had better cell adhesion activity than the commercialized natural human collagen.
  • NIH/3T3 cells were normally cultured and passaged (purchased from the Cell Bank of the Chinese Academy of Sciences, Cat. No. GNM6, and the cultivation and passage methods were carried out according to the instructions of the cells).
  • Cells in good condition were inserted into a 6-well plate, and 2 mL of cell suspension was inoculated in each well at a density of 20,000 cells/mL, and cultured for 36 h.
  • Use a 200 ⁇ L pipette tip to make a scratch, wash the cells 3 times with PBS, and remove the scratched cells.
  • the in vitro cell migration experiment simulates the process of cell migration in vivo to a certain extent, and directly reflects the interaction between cells and extracellular matrix and cells under the influence of matrix.
  • Cell migration activity is a more effective indicator to characterize the biological activity of collagen. The higher the migration rate and the faster the speed, the better the biological activity of collagen.
  • Figure 20 the actual comparison diagram of cell migration taken at different times (in the two black lines are the scratch wound area after the initial and cell migration), and the calculated cell migration rate shown in Figure 21 (Image J Comparing the calculated cell-free blank area), it can be seen that recombinant human type XVII collagen 170801 and 170802 have better cell migration activity than commercial natural human collagen.
  • sodium carboxymethyl cellulose Take sodium carboxymethyl cellulose, respectively take 170801 and 170802 purified freeze-dried collagen products and dissolve them in ultrapure water to obtain a concentration of 0.01% sodium carboxymethyl cellulose and 5% collagen, and 0.01% carboxymethyl fiber Sodium sodium and 5% collagen were mixed to prepare a collagen gel, and sodium carboxymethylcellulose gel without collagen was used as a negative control.
  • New Zealand albino rabbits purchased from the Experimental Animal Center of the Naval Medical University of the Chinese People's Liberation Army), weighing 2-3kg. New Zealand albino rabbits were reared in a well-ventilated room at a room temperature of 21°C, with a 12-hour day-night cycle, in a single cage, and were bred for 2 weeks to adapt to the environment before the experiment. Then the scar model was constructed: the experimental animals received 30 g/L pentobarbital sodium 1.0 mL/kg (30 mg/kg) for ear vein anesthesia. After confirming the anesthesia, wipe and disinfect the skin 3 times with alcohol.
  • a skin biopsy device with a diameter of 1 cm was used to draw a neat skin incision on the inner side of the rabbit ear, and a full-thickness skin defect wound with a diameter of about 1 cm was cut out with ophthalmic scissors, exposing to the cartilage bone surface of the ear. Cover the wound with sterile vaseline oil gauze, and cover the outside with sterile gauze, and bandage and fix it. Disinfect and debride the wound with povidone iodine every 2-3 days, and replace the wound dressing.
  • Collagen gel 170801 and collagen gel 170802 were used to smear the wounds respectively, and iodine-containing antibacterial surgical patch (Drape Minnesota Mining and Manufacturing Company, USA), self-adhesive silicone film dressing ( Smith & Nephew, England) for scar intervention.
  • iodine-containing antibacterial surgical patch Drape Minnesota Mining and Manufacturing Company, USA
  • self-adhesive silicone film dressing Smith & Nephew, England

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Abstract

本发明提供一种重组人XVII型胶原蛋白，所述胶原蛋白，由(A)n所示的氨基酸序列组成或包含(A)n所示的氨基酸序列，其中A为SEQ ID NO.2所示序列或在SEQ ID NO.2基础上进行一定程度上氨基酸修改后的序列或与SEQ ID NO.2具有大于80％同源性的序列；其中n为大于或等于1的整数；其中每个A相同或不同并且以A为基础单元串联重复，每个相同或不同的A之间直接通过肽键相连接。该胶原蛋白可与毕赤酵母中可溶性表达，具有细胞粘附活性、促细胞迁移活性以及组织再生和促进毛囊修复再生的生物学活性，并且可工业化生产。

Description

一种重组人XVII型胶原蛋白、制备方法和应用 技术领域
本发明涉及一种重组人XVII型胶原蛋白、制备方法和应用，属于基因工程技术领域。
背景技术
胶原蛋白作为重要的天然生物蛋白，可广泛应用于化工、医药、食品、化妆品等众多领域，尤其适合制备多种生物器械，是最为理想的生物材料来源，具有广阔的应用前景。市场上销售胶原蛋白主要是利用酸、碱、酶解法处理动物组织获得的胶原提取物。这类提取物在加工过程中，基本破坏了胶原的天然结构，降解严重，使其生物活性丧失；提取的胶原肽长度不等、性质不均、质量不稳定且有病毒感染的风险；同时动物源与人的胶原蛋白的氨基酸序列差别较大，会导致免疫排斥和过敏症状。
人XVII型胶原蛋白是一种跨膜型非成纤维胶原蛋白，其为三条相同α1(XVII)链构成的均相三聚体，分为胞内、跨膜、胞外三大类结构域。人XVII型胶原蛋白是细胞中半桥粒的一种组成成分，在上皮细胞-基底膜的作用中发挥重要作用，可调控上皮细胞的黏附、分离和发育分化，对角化细胞的分化和再生有重要作用。人XVII型胶原蛋白在人体中含量极少，且XVII型胶原蛋白在动物体内含量同样极为稀少，提取难度也非常大，无法量产，同时动物源性胶原蛋白产品不可避免的有免疫原性和潜在的病毒、疫病等生物安全性隐患，没有大量应用。限于法理伦理的约束，人源XVII型胶原蛋白只能应用于科研层面，这些也就导致现在市场上没有人或其它动物来源的商品化XVII型胶原蛋白产品，这也进一步限制了人XVII型胶原蛋白的研究和应用。现在解决此类问题的主要方式则是通过基因工程等生物技术来完成胶原蛋白。
现有重组蛋白主要由原核(大肠杆菌)表达系统，毕赤酵母表达系统、哺乳动物细胞表达系统、昆虫细胞(杆状病毒)表达系统四大类表达系统进行生产。关于哺乳动物细胞表达人XVII型胶原蛋白的研究较少，且均处于实验研究阶段；最早使用原核表达(大肠杆菌、pGEX表达载体)的是非螺旋区NC16序列的融合蛋白，其余表达系统暂无成功表达人XVII型胶原蛋白的报道，尤其是毕赤酵母表达系统。
其中哺乳动物细胞表达成本高、产量低，多用于高价值的药用蛋白表达生产。哺乳动物细胞表达的人XVII型胶原蛋白，无论是瞬时转染表达还是稳定转染细胞系表达，蛋白质表达量极低，同时其所使用的培养基昂贵，只能满足科学研究中微量的使用。昆虫细胞(杆状病毒)表达系统除成本高、成量低外，其翻译后与人细胞差异巨大，胶原蛋白的大量生产应用一般不采用这两种方式。原核表达(大肠杆菌表达系统)的胶原蛋白没有对蛋白质的翻译后修饰，有大规模放大前景的原核表达只能是胞内表达，需要进行菌体的裂解，会有大量杂质 蛋白与目的蛋白混合，对纯化工艺都有极高的要求，且原核表达体系天然带有(细菌细胞壁成分)内毒素、肽聚糖，需要复杂的纯化工艺方能去除。
以上的几个表达系统中，均存在序列的单一性与生物学活性的问题，序列选取比较单一、胶原蛋白肽均是分别独立进行表达等，相对于人XVII型胶原蛋白胞外域接近1000个氨基酸(分15个三螺旋区、15个非三螺旋区)的序列丰富性而言，这两者是欠缺的，这也直接影响了生物学活性，胶原的生物学活性中细胞粘附活性只是最基础的一项，其余的生物学活性均未涉及。并且，无论是以上的哺乳动物细胞表达，还是大肠杆菌表达，均只停留于小体积实验室级别的培养、生产，均没有进行规模化生产。但重组胶原蛋白的应用前提是能够进行规模化、高密度、高表达的发酵生产和纯化，一般来经过说500L体积的中试发酵实验的验证方有工业级别、规模化放大生产的可能性。
人XVII型胶原蛋白表达存在一些难点，人XVII型胶原蛋白属于跨膜类的胶原蛋白，有胞内区、跨膜区、胞外区。一般来说跨膜蛋白于真核细胞中表达时多数时候并不会分泌于胞外，而是会固定于细胞膜上，而且人XVII型胶原蛋白的氨基酸序列很长(1497个氨基酸)、蛋白质分子量较大(180kDa)，理论上很难有效分泌于胞外且易于降解，要成功表达就需要进行相关序列的选取。同时，现有人XVII型胶原蛋白的研究对其氨基酸序列、结构、功能等方面的研究知之甚少。所以，如何选取氨基酸序列使其能在保持毕赤酵母表达体系优点(尤其是分泌表达)的前提下实现更多生物学功能是突出的难点，当然，这一点在其它表达系统中(包括大肠杆菌)表达人XVII型胶原蛋白也是难点。
发明内容
本发明的目的在于，克服现有技术中存在的一些技术问题，主要是克服关于重组人XVII型胶原蛋白序列(非单一性)的优化选取及能否在毕赤酵母高效分泌表达、现有重组人XVII型胶原蛋白仅有细胞黏附活性、现有重组人XVII型胶原蛋白未能规模化制备的生产的技术问题。
为此，本发明提供一种胶原蛋白、编码该胶原蛋白的多核苷酸、含有编码该胶原蛋白的多核苷酸的重组载体、工程菌、制备该胶原蛋白的方法、包含该胶原蛋白的组合物、该胶原蛋白的应用。本发明中已经证实该胶原蛋白不但能够实现人XVII型胶原蛋白于毕赤酵母这种真核宿主细胞的高效分泌可溶性表达，而且还具有比商品化的天然人胶原蛋白更为优异的细胞粘附活性、促细胞迁移活性以及优异组织再生和促进毛囊修复再生的生物学活性，并且可工业化应用。
本发明提供了一种胶原蛋白，所述胶原蛋白具有细胞粘附活性、促细胞迁移活性、促进组织再生和促进毛囊修复再生的生物学活性。
所述胶原蛋白，由(A) n所示的氨基酸序列组成或包含(A) n所示的氨基酸序列，其中：A为SEQ ID NO.2所示的氨基酸序列或在SEQ ID NO.2基础上进行一定程度上的氨基酸替换、插入、取代、添加、缺失等修改后的氨基酸序列或与SEQ ID NO.2氨基酸序列具有大于80％同源性的氨基酸序列；其中n为大于或等于1的整数；其中每个A相同或不同并且以A为基础单元串联重复，每个相同或不同的A之间直接通过肽键相连接。
进一步的，根据本发明的一个实施例，所述胶原蛋白由(A) n所示的氨基酸序列组成或包含(A) n所示的氨基酸序列：其中A为SEQ ID NO.2所示的氨基酸序列；n为大于或等于1的整数；其中每个A相同且以A为基础单元串联重复，每个A之间直接通过肽键相连接。
进一步的，根据本发明的一个实施例，所述胶原蛋白由(A) n所示的氨基酸序列组成，当n为1时，所述胶原蛋白的氨基酸序列如SEQ ID NO.2所示，本发明中记为170801；当n为2时，所述胶原蛋白的氨基酸序列如SEQ ID NO.3所示，本发明记为170802。
进一步的，根据本发明的记载，本发明所述胶原蛋白由(A) n所示的氨基酸序列组成或包含(A) n所示的氨基酸序列，其中A为在SEQ ID NO.2基础上进行一定程度上的氨基酸插入、取代、添加、缺失等修改后的氨基酸序列或与SEQ ID NO.2氨基酸序列具有大于80％同源性的氨基酸序列；其中n为大于或等于1的整数；其中每个A相同或不同并且以A为基础单元串联重复，每个相同或不同的A之间直接通过肽键相连接。
本发明还提供编码上述胶原蛋白的多核苷酸，具体为编码上述(A) n代表的胶原蛋白的多核苷酸序列；优选的，以毕赤酵母为宿主细胞时，所述多核苷酸为包含SEQ ID NO.6或SEQ ID NO.7所示的核苷酸序列或由SEQ ID NO.6、SEQ ID NO.7所示的核苷酸序列组成。
根据本发明的一个实施例，所述A为SEQ ID NO.2，n为1时的多核苷酸为由SEQ ID NO.6所示的核苷酸序列；根据本发明的一个实施例，所述A为SEQ ID NO.2，当n为2时，编码上述(A)n的多核苷酸为由SEQ ID NO.7所示的核苷酸序列。
本发明的胶原蛋白，是从人XVII型胶原蛋白的第15螺旋区、羧基端区、中间区域等多个螺旋区序列中优化筛选后选取的组合序列，与人XVII型胶原蛋白氨基酸序列相应部分100％相同，不但能够实现人XVII型胶原蛋白于毕赤酵母这种真核宿主细胞的高效分泌可溶性表达，而且还具有比商品化天然人胶原蛋白更为优异的细胞粘附活性、促细胞迁移活性以及促进组织再生和促进毛囊修复再生的生物学活性；这样的组合序使各区域序列的功能得以集成，并不是分别单独表达而是作为一个整体进行表达，避免了重组胶原蛋白的序列单一性。
本发明还提供了含有编码上述胶原蛋白的多核苷酸的重组表达载体。
根据本发明的一个实施例，本发明所述的重组表达载体构建过程中，首先对编码上述(A) n代表的胶原蛋白的多核苷酸进行修饰，分别于胶原蛋白氨基酸序列的氨基端添加编码 Strep-Tag II标签的DNA序列、羧基端添加编码6×His Tag标签的DNA序列，使其含有双特异性亲和纯化标记，这样可进行亲和层析纯化，也便于以两种标签序列为基础进行免疫学抗体检测。将标记后的序列连接入表达载体pPIC9K中，构建重组表达载体(实施例中表示为pPIC9K-170801、pPIC9K-170802)。
本发明还提供由上述重组表达载体构建的工程菌，所述工程菌的宿主细胞优选为毕赤酵母。所述工程菌保藏日期为2020年09月09日，保藏编号分别为CGMCC NO.20626、CGMCC NO.20627，分类命名为巴斯德毕赤酵母Pichia pastoris，保藏单位为中国微生物菌种保藏管理委员会普通微生物中心，地址为北京市朝阳区北辰西路1号院3号。
需要注意的是，宿主细胞可以是真核细胞，例如真菌、酵母、植物细胞等；原核细胞，例如肠杆菌科细菌，如大肠杆菌；也可以是动物细胞，例如哺乳动物细胞CHO系列细胞、HEK293系列细胞或昆虫细胞。应当理解，本领域技术人员可以通过用其它表达宿主代替上述毕赤酵母作为表达宿主表达出相同氨基酸序列的胶原蛋白。
本发明还提供上述胶原蛋白的制备方法，包括如下步骤：
(1)工程菌的培养和筛选
将构建的工程菌经过筛选后，接入BMGY培养基诱导表达筛选表达高的工程菌。所述筛选的表达量高的工程菌为巴斯德毕赤酵母(Pichia pastoris)，保藏编号为CGMCC No.20626或CGMCC No.20627。
(2)规模化高密度发酵、培养和蛋白质纯化
采用50L-500L发酵罐进行联动分批补料发酵，对发酵上清液进行纯化，获取高纯度胶原蛋白。
根据本发明的一个实施例，本发明以Sac I线性化重组表达载体，电转入毕赤酵母感受态细胞，构建工程菌，将工程菌转涂至MD平板初筛后，再经过含有不同浓度G418的YPD平板筛选，挑取菌落接入BMGY培养基中，再以BMMY培养基诱导表达；挑取多株菌种，选择表达量高的工程菌进行后续实验。本发明中，以SDS-PAGE电泳、Western Blot对表达的蛋白质进行初步鉴定，判断其有效分泌表达的能力，结果证明了本发明的重组人XVII型胶原蛋白能够高效分泌表达，而全长的人XVII型胶原蛋白主要集中于胞内无法有效分泌于胞外；将电泳获得的目标条带进行Nano-HPLC-MS/MS质谱检测，证明其中肽段来源于人XVII型胶原蛋白序列中优化选取的区域，本发明的重组人XVII型胶原蛋白170801、170802成功表达。
采用50L-500L发酵罐诱导表达48h时，本发明的胶原蛋白产量(UV定量)可达到12g/L、11g/L。
本发明中，使用MALDI-TOF/TOF进行分子量的测定，170801胶原蛋白的分子量为23811.6797Da(理论值为23800Da)、170802胶原蛋白的分子量为45689.5781Da(理论值为45400Da)，毕赤酵母表达后存在翻译后修饰及分子量实验测定存在的系统误差，实际表达的胶原蛋白分子量与理论值一致。使用对胶原蛋白红外光谱扫描，其酰胺A、酰胺B、酰胺I、酰胺II、酰胺III的波数均符合重组胶原蛋白结构特征。电镜扫描纯化的胶原蛋白冻干海绵，有片层状结构，有应用于生物医学材料领域的潜力。
根据本发明的一个实施例，对得到的胶原蛋白的生物学活性检测，体外细胞实验验证其细胞粘附活性、促细胞迁移活性。对得到的胶原蛋白生物学活性的动物实验，兔耳瘢痕模型实验验证其能够有效的促进兔耳瘢痕的愈合、组织再生，尤其是有极为优异的促进毛囊修复再生的活性。
基于此，本发明还提供上述胶原蛋白在促进组织再生、促进毛囊修复方面的用途。
本发明还提供一种组合物，所述组合物包含上述胶原蛋白或上述方法制备的胶原蛋白。
本发明还提供一种制品，所述制品包含上述胶原蛋白或上述方法制备的胶原蛋白或上述组合物。所述制品包括但不局限于药物、药物组合物、医疗器材、生物材料、组织工程产品、化妆品或保健品等。
本发明还提供所述胶原蛋白、核苷酸、重组表达载体、工程菌、组合物等在制备制成品中的用途，包括但不限于药物、药物组合物、医疗器材、生物材料、组织工程产品、化妆品、保健品中的用途。
本发明还提供所述胶原蛋白、核苷酸、重组表达载体、工程菌、或组合物在制备促进瘢痕愈合、组织修复或促进毛囊修复的产品中的用途。根据本发明的一个实施例，所述产品为外用制剂，优选为外用涂抹制剂，优选的为凝胶剂。
本发明的有益效果：
(1)本发明选择了毕赤酵母表达系统，毕赤酵母是真核微生物，拥有真核生物完整的细胞器，能对翻译的蛋白质进行一定的翻译后修饰(尤其是糖基化修饰)，有力支撑蛋白质生物学功能的实现。以其建立表达系统同时拥有微生物表达系统可高密度发酵生产、极低的培养生成本、短周期、高表达等规模化工业生产的优点；其可分泌于胞外的特性，可完全菌体裂解带来的杂质蛋白；细胞壁成分中不含内毒素、肽聚糖。
(2)本发明中重组人XVII型胶原蛋白选取的序列为经过筛选优化的序列组合而成，其氨基酸序列与天然胶原蛋白氨基酸序列相应部分同源性为100％，无免疫原性，可以广泛应用于医药、医疗器械、生物材料、组织工程、化妆品等领域。
(3)本发明中重组人XVII型胶原蛋白经过实验验证，具有与商品化的天然人胶原相同 或更优的细胞粘附活性、促细胞迁移活性、组织修复和促进毛囊修复再生等多种生物学活性，能达到真正产品应用的目的。
(4)本发明中重组人XVII型胶原蛋白可于毕赤酵母表达系统中高效分泌表达，易于纯化获得高纯度的胶原蛋白；编码氨基酸序列的基因DNA序列进行了优化设计，使得构建的工程菌株蛋白表达量很高，可于高密度发酵表达时获得很高的产量；毕赤酵母易于进行高密度发酵表达，本发明筛选的菌种已能实现500L级中试规模的高密度高表达量发酵，已具备工业化广大生产的条件。
(5)本发明中的宿主细胞毕赤酵母对表达的外源蛋白有一定的翻译后修饰，例如糖基化等。
附图说明
图1为本发明构建的pPIC9K-170801载体图谱。
图2为本发明构建的pPIC9K-170802载体图谱。
图3为本发明构建的pPIC9K-col17a1载体图谱。
图4为重组全长人XVII型胶原蛋白α1链表达的SDS-PAGE结果图，其表观分子量约为180kDa。
图5为重组全长人XVII型胶原蛋白α1链使用抗His标签抗体进行Western Blot检测结果。
图6为诱导24h后170801、170802胶原蛋白的工程菌表达上清的SDS-PAGE结果。
图7为诱导24h后170801、170802胶原蛋白的工程菌表达上清使用抗His标签抗体进行Western Blot检测结果。
图8为诱导24h后170801、170802胶原蛋白的工程菌表达上清使用抗Strep-Tag II标签抗体进行Western Blot检测结果。
图9为170801胶原蛋白的SDS-PAGE条带的质谱分析与天然人XVII型胶原蛋白序列比对结果。
图10为170802胶原蛋白的SDS-PAGE条带的质谱分析与天然人XVII型胶原蛋白序列比对结果。
图11为171801胶原蛋白的工程菌在500L发酵罐中试发酵菌生长曲线和胶原蛋白表达曲线。
图12为171802胶原蛋白的工程菌在500L发酵罐中试发酵菌生长曲线和胶原蛋白表达曲线。
图13为纯化后的170801胶原蛋白HPLC图谱。
图14为纯化后的170802胶原蛋白HPLC图谱。
图15为170801胶原蛋白分子量的MALDI-TOF MS检测结果。
图16为170802胶原蛋白分子量的MALDI-TOF MS检测结果。
图17为纯化后的170801、170802胶原蛋白的红外图谱。
图18为170801(左)、170802(右)胶原蛋白冻干海绵样品的SEM图(200×)。
图19为170801、170802胶原蛋白的细胞粘附活性检测结果。
图20为170801、170802胶原蛋白与天然人胶原蛋白的细胞迁移状态实际对比结果。
图21为细胞迁移24h、48h后，170801、170802胶原蛋白与天然人胶原蛋白的细胞迁移率。
图22为170801、170802胶原蛋白对兔耳瘢痕干预6周的瘢痕大体照片和瘢痕切片图。
具体实施方式
为了使本领域技术人员更好的理解本发明的技术方案，下面对本发明的较佳实施例进行详细的阐述，但是如下实施例并不限制本发明的保护范围。
本发明的实施例中，没有多作说明的都是采用常规分子生物学实验方法完成，实施例中所涉及PCR、酶切、连接、密码子优化等过程都是本领域技术人员根据产品说明书或本领域基础知识可以理解并且容易实现的，因此不再详细描述。
实施例1.重组人XVII型胶原蛋白的构建、表达和鉴定
(1)重组人XVII型胶原蛋白氨基酸序列的设计
本发明中，基于人XVII型胶原蛋白序列优化，所述人XVII型胶原蛋白具体序列参考：Uniprot Q9UMD9-1序列(https://www.uniprot.org/uniprot/Q9UMD9)和NCBI参照序列Q9UMD9.3(https://www.ncbi.nlm.nih.gov/protein/Q9UMD9.3)，二者序列相同，具体如SEQ ID NO.1所示。
SEQ ID NO.1：
Figure PCTCN2022071968-appb-000001
Figure PCTCN2022071968-appb-000002
上述序列SEQ ID NO.1中加粗有下划线部分即为本发明所选取的序列，共233个氨基酸。本发明所选取的序列是从人XVII型胶原蛋白的第15螺旋区、羧基端区、中间区域等多个螺旋区序列中优化筛选后选取的组合序列，不但能够实现人XVII型胶原蛋白在毕赤酵母这种真核宿主细胞的高效分泌可溶性表达，而且还具有比商品化的天然人胶原蛋白更为优异的细胞粘附活性、细胞迁移活性以及优异组织再生和促进毛囊修复再生的生物学活性；这样的组合使各区域序列的功能得以集成，并不是分别单独表达而是作为一个整体进行表达，避免了重组胶原蛋白的序列单一性，具有极强的创新性，本发明中将其被命名为170801，是人XVII型胶原蛋白的优选序列，具体序列如SEQ ID NO.2所示。
SEQ ID NO.2：
Figure PCTCN2022071968-appb-000003
编码170801的DNA序列如SEQ ID NO.6所示：
SEQ ID NO.6：
Figure PCTCN2022071968-appb-000004
Figure PCTCN2022071968-appb-000005
本发明中的胶原蛋白，还包括以170801为基本单位，进行n次串联重复，得到的氨基酸序列组成，其中n为大于或等于1的整数。
当以170801为基本单位，当n为2时，170801进行2次串联重复，得到的氨基酸序列，共466个氨基酸，将其命名记为170802，其氨基酸序列如SEQ ID NO.3所示。
SEQ ID NO.3：
Figure PCTCN2022071968-appb-000006
编码170802的DNA序列如SEQ ID NO.7所示。
SEQ ID NO.7：
Figure PCTCN2022071968-appb-000007
Figure PCTCN2022071968-appb-000008
蛋白质的功能需要其氨基酸残基有序的排列方能实现，本发明中重组人XVII型胶原蛋白所有创新性生物学活性建立在优化筛选选取的氨基酸序列的基础之上，有限的序列修改或一定比例的同源性(大于80％)仍可能实现相同或类似的生物学活性。
所以本发明中，所述的胶原蛋白，还包括在SEQ ID NO.2基础上进行一定程度上的氨基酸插入、取代、添加、缺失等修改后的氨基酸序列或与SEQ ID NO.2氨基酸序列具有大于80％同源性的氨基酸序列，以该氨基酸序列为基本单元；进行n次串联重复得到的氨基酸序列。其中n为大于或等于1的整数，每个基本单元相同或不同。
其中，本发明所述的同源性指的是序列的重合率，可以是两序列直接的数量关系，如部分相同、相似的百分比；还包括两序列在同一位点核苷酸或氨基酸残基完全相同的序列比例；也可以是从某个共同祖先经趋异进化形成的不同的序列。可以通过常规生物信息学方法比对序列信息得到。
所述的氨基酸插入指在氨基酸序列，例如SEQ ID NO.2或SEQ ID NO.3序列的适当位置插入氨基酸残基，插入的氨基酸残基也可以全部或部分彼此相邻，或插入的氨基酸之间都不彼此相邻。只要所述蛋白具有与170801、170802胶原蛋白有类似的生物学活性，包括细胞粘附活性、促细胞迁移活性、促进组织再生和促进毛囊修复再生的活性。
所述的氨基酸取代指在氨基酸序列，例如SEQ ID NO.2或SEQ ID NO.3序列某个位置的某个或若干个氨基酸残基(包括连续若干个氨基酸残基或非连续的若干个氨基酸残基)被其他氨基酸残基替代，只要所述蛋白具有与170801、170802胶原蛋白有类似的生物学活性， 包括细胞粘附活性、促细胞迁移活性、促进组织再生和促进毛囊修复再生的活性。
所述的氨基酸添加指在氨基酸序列，例如SEQ ID NO.2或SEQ ID NO.3序列的C端或N端添加氨基酸，只要所述蛋白具有与170801、170802胶原蛋白有类似的生物学活性，包括细胞粘附活性、促细胞迁移活性、促进组织再生和促进毛囊修复再生的活性。
所述的氨基酸缺失指可以从氨基酸序列，例如SEQ ID NO.2或SEQ ID NO.3序列中删除1、2或3个及3个以上氨基酸，只要所述蛋白具有与170801、170802胶原蛋白有类似的生物学活性，包括细胞粘附活性、促细胞迁移活性、促进组织再生和促进毛囊修复再生的活性。
本发明中所述的氨基酸优选为L型氨基酸；同时其中的一个或多个(如2-5个、2-4个或2-3个)氨基酸也可以用构象为D型的氨基酸、人工修饰的氨基酸、自然界存在的稀有氨基酸等进行替换，以提高多肽的生物利用度、稳定性和/或抗病毒活性。其中D型氨基酸是指与组成蛋白质的L型氨基酸相对应的氨基酸；人工修饰的氨基酸指经过甲基化、磷酸化等修饰的组成蛋白质的常见L型氨基酸；自然界存在的稀有氨基酸包括组成蛋白质的不常见氨基酸和不组成蛋白质的氨基酸，例如5-羟基赖氨酸、甲基组氨酸、γ氨基丁酸、高丝氨酸等。
(2)重组人XVII型胶原蛋白氨基酸序列和DNA序列优化
本发明中，对170801、170802的DNA序列进行修饰，分别于二者氨基端添加编码Strep-Tag II标签的DNA序列、羧基端添加编码6×His Tag标签的DNA序列，使其含有双特异性亲和纯化标记，这样可进行亲和层析纯化，也便于以两种标签序列为基础进行免疫学抗体检测。具体的是以170801、170802的氨基酸序列及其基因序列为基础，进行DNA序列的密码子偏好性及其转录、翻译过程中相关优化参数进行计算、拼接重组和优化，合成更适宜在毕赤酵母中高效表达的DNA序列。
本发明中，在170801两端分别修饰，氨基端添加编码Strep-Tag II标签的DNA序列、羧基端添加编码6×His Tag标签的DNA序列后，170801最终表达获得的是含有标签的蛋白，共249个氨基酸，优化后的170801的氨基酸序列如SEQ ID NO.4所示。在170801两端分别修饰，氨基端添加编码Strep-Tag II标签的DNA序列、羧基端添加编码6×His Tag标签的DNA序列、终止密码子(TAA)及Not I的酶切位点的DNA序列如SEQ ID NO.8所示。
SEQ ID NO.4：
Figure PCTCN2022071968-appb-000009
SEQ ID NO.8：
Figure PCTCN2022071968-appb-000010
同样的，在170802两端分别修饰添加氨基端添加编码Strep-Tag II标签的DNA序列、羧基端添加编码6×His Tag标签的DNA序列后，170802最终表达获得的是含有标签的蛋白，共482个氨基酸，优化后的170802的氨基酸序列如SEQ ID NO.5所示。在170802两端分别修饰添加氨基端添加编码Strep-Tag II标签的DNA序列、羧基端添加编码6×His Tag标签的DNA序列和终止密码子(TAA)及Not I的酶切位点的DNA序列如SEQ ID NO.9所示。
SEQ ID NO.5：
Figure PCTCN2022071968-appb-000011
SEQ ID NO.9：
Figure PCTCN2022071968-appb-000012
Figure PCTCN2022071968-appb-000013
本发明中，编码全长人XVII型胶原蛋白氨基酸序列(即人XVII型胶原蛋白α1链)的DNA序列(氨基端添加EcoR I的酶切位点、Strep-Tag II标签的DNA序列、羧基端添加编码6×His Tag标签的DNA序列和终止密码子(TAA)及Not I的酶切位点)如SEQ ID NO.10所示。
SEQ ID NO.10：
Figure PCTCN2022071968-appb-000014
Figure PCTCN2022071968-appb-000015
Figure PCTCN2022071968-appb-000016
(3)DNA序列的合成与重组表达载体的构建
本发明中，优化后的序列SEQ ID NO.8、SEQ ID NO.9和SEQ ID NO.10委托南京金斯瑞生物科技股分有限公司合成，三个基因片段记为pUC57-170801、pUC57-170802、pUC57-col17a1。将合成后的基因片段SEQ ID NO.8、SEQ ID NO.9使用SnaB I和Not I双酶切，将酶切后得到的目的基因片段连接入同样双酶切后的pPIC9K空载体(购自赛默飞世尔科技公司)。同时将合成后的基因片段SEQ ID NO.10使用EcoR I和Not I双酶切，将酶切后得到的目的片段连接入同样双酶切后pPIC9K空载体，作为对比。
步骤a.扩增目的基因、表达载体：以质粒小提试剂盒(购自生工生物工程(上海)股份有限公司)分别提取pUC57-170801、pUC57-170802、pUC57-col17a1、pPIC9K空载体的质粒，具体操作按试剂盒说明书进行。
步骤b.将步骤a得到的目的基因片段与pPIC9K载体的质粒在37℃下双酶切30min，酶切反应体系如下(所用QuickCut TM限制性酶购自大连TaKaRa公司，酶切过程按其说明书进行)：
质粒 3μg
10×H缓冲液 5μL
EcolRⅠ/SnaB I 10U
NotⅠ 10U
无菌水 至50μL
将所得到的目的基因片段和载体片段，采用PCR产物纯化试剂盒进行纯化(购自大连TaKaRa公司，具体操作按试剂盒说明书进行)。回收经双酶切处理后的目的基因片段和载体pPIC9K线性化片段，用SolutionⅠ连接试剂(连接试剂盒购自大连TaKaRa公司)进行连接(16℃连接30min)，将目的基因片段准确插入到含有分泌信号α-因子的分泌型载体读码框内，连接反应体系如下(按连接试剂盒说明书进行)：
pPIC9K线性化片段 2μL
目的基因片段 3μL
SolutionⅠ连接试剂 5μL
将连接产物转化进入感受态大肠杆菌DH5α(购自生工生物工程(上海)股份有限公司)，在含有Amp的LB抗性平板筛选阳性克隆，进行菌落PCR验证，提取重组质粒进行测序鉴定(交由生工生物工程(上海)股份有限公司完成)，测序验证正确的证明本实施例成功构建了重组表达载体pPIC9K-170801、pPIC9K-170802、pPIC9K-col17a1，相关质粒图谱如图1、图2和图 3所示。
(4)重组工程菌株的构建、菌种筛选
分别将步骤(3)得到的重组表达载体质粒10μg用SacⅠ(酶切试剂盒购自大连TaKaRa公司，具体操作按试剂盒说明书进行)37℃酶切消化过夜，使其线性化，再使用以PCR产物纯化试剂盒(购自生工生物工程(上海)股份有限公司)，回收线性化质粒，回收的体积控制在10μL左右。
将线性化质粒电转化入空宿主菌种毕赤酵母GS115(购自中国工业微生物菌种保藏管理中心)感受态细胞中，将电转后的菌液涂布于MD平板上，每100μL～200μL涂布一块平板，室温静置10min，于30℃倒置培养2-5天，直至有单菌落(阳性转化子)出现。
向MD平板表面加入2mL无菌双蒸水，然后用无菌三角涂布器轻轻刮下平板表面的His +转化子(阳性转化子)，并转移到50mL离心管中，以无菌双蒸水稀释成菌悬液，含10 5个细胞的菌悬液涂布于含有0.5mg/mL G418的YPD平板上，倒置，30℃培养3～4d后至单菌落出现。从YPD平板上挑取菌落至无菌96孔板中(每孔含200μL YPD)，混匀，于30℃培养48h；混匀孔中菌液，各取10μL接入至一块新的无菌96孔板，于30℃培养24h后再重复一次该步骤；24h后，从第三块96孔板中取出1μL分别点在含有1.0mg/mL和4mg/mL G418的YPD平板上，于30℃继续培养96h～120h。毕赤酵母转化子若能在含高浓度G418的平板上生长，说明该转化子含有多拷贝的目的基因，即有多个重组片段进入了酵母体内并通过同源重组整合到酵母的染色体上。经过这一步筛选可得到的高拷贝、可高效表达的重组酵母工程菌种。
构建分别含pPIC9K-col17a1、pPIC9K-170801、pPIC9K-170802三种工程菌样本均送至中国微生物菌种保藏管理委员会普通微生物中心，菌种保藏编号分别是：CGMCC No.18659、CGMCC No.20626、CGMCC No.20627。地址：北京市朝阳区北辰西路1号院3号；保藏日期：CGMCC No.18659为2019年10月10日、CGMCC No.20626、CGMCC No.20627为2020年09月09日。分类命名：巴斯德毕赤酵母Pichia pastoris。
(5)诱导表达与重组胶原蛋白的鉴定
在步骤(4)中含有1.0mg/mL和4mg/mL G418的YPD平板上分别挑选单菌落，置于装有10mL BMGY培养基的100mL三角瓶中，于28-30℃、220rpm培养至OD 600为2～6(16-18h)。室温下1500～3000g离心5min，收集菌体，用BMMY培养基重悬菌体，使OD 600为2左右，放置于28-30℃、220rpm的摇床上继续生长3天，每24h向培养基中添加100％甲醇至终浓度为1.0％。按时间点(≥24h，必要时24h、48h、72h、96h均取样)分别取菌液样品，取样量为1mL，置于1.5mL EP管中，最大转速离心2～3min，收集表达上清和菌体。将菌体以玻璃株(购自生工生物(上海)有限公司)破碎法裂解，裂解缓冲液(1mM PMSF、10mM Tris， pH7.4)，4℃下以12000g离心5min，取胞内裂解上清，再加入8M尿素振荡溶解沉淀，分析目的蛋白的表达量和菌液最佳收获时间。待检测样品于-80℃保存备用。
收取的表达上清、胞内裂解上清、胞内沉淀溶解液，加入5×上样缓冲液(250mM Tris-HCl、pH值为6.8，10％SDS，0.5％溴酚蓝，50％甘油，5％β-巯基乙醇)，置于100℃金属浴加热10min，进行SDS-PAGE检测。因表达的目的蛋白氨基端有Srtep-TagⅡ标签，羧基端有6×His标签，可以抗Srtep-TagⅡ、抗6×His Tag的抗体(购自南京金斯瑞生物科技股份有限公司)，采用Western Blot进行检测。
重组全长人XVII型胶原蛋白α1链的表达与鉴定：
如图4的SDS-PAGE结果图所示，表达上清中并没有可见预期的目的条带(其表观分子量约为180kDa)。
使用抗6×His Tag抗体进行Western Blot检测，结果如图5所示，发现表达上清中只能检测到极少量降解后的目的蛋白(表观分子量小于预期，约为120kDa)，并且是在诱导表达很长时间后方能看到；胞内裂解上清中有较多的降解后的目的蛋白(表观分子量小于预期)，其表观分子量约为120kDa(与人XVII型胶原蛋白胞外域的表观分子量一致)；只有胞内沉淀溶解部分才有预期表观分子量大小(180kDa)的目的蛋白存在。实验结果说明：作为一种跨膜类型的胶原蛋白，其应该被固定于胞内的膜系统之上，只有降解后才能脱落溶解于胞内，而表达上清中检测到的降解后目的蛋白极有可能是在培养长时间中已死亡的细胞裂解后释放到表达上清液中的，但无论哪种可能，全长的重组人XVII型胶原蛋白是无法有效分泌于毕赤酵母胞外的。
170801、170802胶原蛋白的表达与鉴定：
图6为诱导24h后，170801、170802胶原蛋白的工程菌表达上清的SDS-PAGE结果，如图6所示，170801、170802胶原蛋白在诱导表达24h即可高效分泌表达于胞外的培养上清中，170801的表观分子量约为30kDa，170802的表观分子量约为52kDa。
图7为诱导24h后，170801、170802胶原蛋白的工程菌表达上清的Western Blot结果(Anti-His，抗体购自生工生物工程(上海)股份有限公司，小鼠抗His单克隆抗体，货号D199987)，图8为诱导24h后，170801、170802胶原蛋白的工程菌表达上清的Western Blot结果(Anti-Strep-Tag II，抗体购自南京金斯瑞生物科技股份有限公司，兔抗Strep-Tag多克隆抗体，货号A00626)。从图7和图8的结果(ECL化学发光显色，全自动化学发光图像分析系统Tanon5200将蛋白质分子质量标准合成于图像)中可以看到，氨基端Srtep-TagⅡ标签，羧基端6×His标签均可检测到，且目的条带大小均与SDS-PAGE中表观分子量大小相同，说明重组人XVII型胶原蛋白170801、170802成功进行了高效分泌表达。
回收切割诱导表达的蛋白条带，用胰蛋白酶将其酶解，Nano-HPLC-MS/MS质谱检测重组胶原蛋白的胰蛋白酶解后肽段(委托苏州普泰生物技术有限公司完成)，并将检测到的肽段进行序列比对(Uniprot数据库)。结果如图9、图10所示：170801、170802胶原蛋白被酶解后检测到的肽段均属于氨基酸序列选取设计时选择的人XVII型胶原蛋白序列的相关区域，说明170801、170802胶原蛋白成功表达。
实施例2.基因工程菌的中试发酵和蛋白纯化
(1)中试发酵
对构建的分别含pPIC9K-170801、pPIC9K-170802的工程菌进行50L-500L联动中试发酵，获取含有重组人XVII型胶原蛋白的发酵液，实现重组人XVII型胶原蛋白170801、170802的规模化表达生产。
种子培养基YPG(配方为：酵母粉10g/L、酵母蛋白胨FP102 20g/L、无水甘油10g/L)；发酵培养基(配方为：NH 4H 2PO 4 190.4g/L、KH 2PO 4 10.06g/L、CaSO 4·2H 2O 1.18g/L、K 2SO 418.2g/L、MgSO 4·7H 2O 14.9g/L、甘油40g/L)；补料培养基(50％W/V甘油，每升加12mL PTM 1微量元素)；诱导培养基(100％甲醇，每升加入12mL PTM 1微量元素)；其中PTM 1用0.22μm的滤膜过滤除菌，4℃保存。发酵培养基高温灭菌后待温度降至室温加入PTM 1，用氨水调节pH值至5.0。
构建的工程菌株分批培养条件和诱导表达条件为：
采用分批补料培养方法，培养温度30℃。将构建的工程菌接入装有种子培养基YPG的摇瓶中，药瓶容量为2L，220rpm、30℃培养24h；以10％接种量接种到含发酵培养基的种子发酵罐中(使其接种后OD 600为5)，发酵罐容量为50L，扩大培养，调节搅拌转速为200r/min-600r/min，空气通量为2VVM，DO≥于30％，培养至碳源耗尽，移种至含发酵培养基的发酵罐中开始发酵培养，发酵罐容量为500L。发酵培养条件为，搅拌转速150r/min-500r/min，罐压为0-0.05MPa，空气通量为1VVM，DO≥于30％，溶氧反弹流加补料培养基，流加速率维持DO≥30％，至OD 600生长到190-210，停止补料。待溶氧反弹DO≥70％后，流加诱导培养基进行诱导表达，调节转速、通气量、罐压和流加速度使DO≥30％。诱导40～72h，UV测量蛋白浓度增长幅度不明显或下降即可放罐。
UV蛋白定量公式：C(mg/mL)＝0.144*(A215-A225)，A215<1.5。
经过对构建的工程菌进行高密度中试发酵，结果发现，诱导48h时，菌浓度(OD 600)均可达到250以上，菌湿重可达到300g/L左右，发酵液中蛋白浓度均大于10g/L(UV定量)。结果见图11和图12，图11可见，171801的工程菌生长良好，诱导48h时菌浓度(OD 600)可达到270，菌湿重可达到355g/L，发酵液中蛋白浓度可达到12g/L(UV定量)。图12可见， 171802的工程菌生长良好，诱导48h时菌浓度(OD600)可达到250，菌湿重可达到293g/L，发酵液中蛋白浓度可达到11g/L(UV定量)。
(2)纯化
本实施例中用到的缓冲液如下：
缓冲液A：20mM KH 2PO 4、pH值为4.0；
缓冲液B：20mM KH 2PO 4、1M NaCl，pH值为4.0。
收集得到的工程菌的发酵液，使用陶瓷膜分离系统(江苏久吾高科技股份有限公司，JWCMF-9)分离菌体和发酵上清。以缓冲液A平衡阳离子交换介质(层析填料为苏州纳微产UniGel-80sp，装载于江苏汉邦科技产全自动层析系统)至A215吸光值和电导率值都保持不变后，设置上样流速为100us/cm，上样体积20L/次，检测紫外A215吸光值，当其上升时，开始接样。待上样结束后，关闭接样，再以缓冲液A平衡阳离子层析介质，当A215吸光值下降时，打开接样，直至紫外和电导降至最低且不再变化，停止接样。分别用500mM和1M的氯化钠洗脱，收集洗脱液，分别检测500mM洗脱液和1M洗脱液组分中纯化蛋白状况，确定好组份后，进行透析(透析液为超纯水)，随后浓缩、冷冻干燥，收集冻干胶原蛋白海绵。
取170801、170802胶原蛋白冻干海绵用超纯水溶解至2mg/mL，0.22μm滤膜过滤，进样10μL(Sepax Bio-C18色谱柱，高效液相色谱仪为Waters2695或Agilent LC1260)，以分析纯化后胶原蛋白170801、170802的纯度。
纯化结果如图13、图14所示，图中可见，纯化的170801、170802胶原蛋白，单峰明显，纯度高(91.2％、90％)，说明本纯化方法可靠有效，得到的胶原蛋白纯度高。
除本实施例所用纯化方法外，因170801、170802胶原蛋白氨基端有Srtep-TagⅡ标签，羧基端有6×His标签，均可使用Ni-NTA、Strep-Tactin等亲和介质进行单种或双亲和纯化，也能达到与本实例类似的效果。
(3)重组胶原蛋白的性质表征
分子量大小测定：
得到的170801、170802蛋白理论预测的分子量分别为23.8kDa、45.4kDa，而SDS-PAGE上检测的表观分子量比理论预测值要大。对重组人XVII型胶原蛋白170801、170802蛋白纯化后冻干品进行基质辅助激光解吸电离飞行时间质谱(AB SCIEX 5800 MALDI-TOF/TOF)检测其相对分子量，判断其蛋白的实际分子量，委托生工生物工程(上海)股份有限公司检测。结果如图15、图16所示，MALDI-TOF MS(电荷数+1)检测到的170801胶原蛋白的分子量为23811.6797Da(理论值为23800Da)、170802胶原蛋白的分子量为45689.5781Da(理 论值为45400Da)，由于毕赤酵母表达后存在翻译后修饰及分子量实验测定存在的系统误差，实际表达的胶原蛋白分子量与理论值一致。
傅里叶变换红外光谱(FT-IR)分析：
胶原蛋白基团的特征吸收峰可以被红外光谱分析检测，试验取微量170801、170802胶原蛋白纯化后冻干样品分别KBr研磨成粉后压片，室温下，在4000～400cm -1范围内扫描(Thermo Scientific，Nicolet TM iS TM 10FT-IR光谱仪)，方法及结果分析参照(Jeong,H.,J.Venkatesan and S.Kim,Isolation and characterization of collagen from marine fish(Thunnus obesus).Biotechnology and Bioprocess Engineering,2013.18(6):p.1185-1191.)
从170801、170802胶原蛋白纯化样品的红外光谱扫描图17可以看到其酰胺A、酰胺B、酰胺I、酰胺II、酰胺III的波数均符合重组胶原蛋白结构特征(参见文献[1].陈静涛等,重组胶原蛋白与牛源Ⅰ型胶原蛋白红外光谱研究.材料导报,2008(03):第119-121页.[2].Doyle,B.B.,E.G.Bendit and E.R.Blout,Infrared spectroscopy of collagen and collagen‐like polypeptides.Biopolymers,1975.14(5):p.937-957.[3].周爱梅等,重组人源胶原蛋白的分离纯化及其结构表征.食品与发酵工业,2015(03):第46-52页.)。
胶原蛋白冻干海绵样品扫描电镜扫描分析：
使用扫描电镜(日立TM3030PLUS)对170801、170802胶原蛋白冻干海绵样品表面进行扫描，结果如图18所示，图中可见，重组人XVII型胶原蛋白170801、170802均具有明显片层状结构，与人体中人XVII型胶原蛋白在基底膜上层状分布类似，这样的结构预示其具有应用于生物医学材料领域的潜力。
实施例3.重组胶原蛋白细胞黏附活性检测
重组胶原蛋白的细胞黏附活检测方法参考文献Juming Yao,Satoshi Yanagisawa,Tetsuo Asakura.Design,Expression and Characterization of Collagen-Like Proteins Based on the Cell Adhesive and Crosslinking Sequences Derived from Native Collagens,J Biochem.136,643-649(2004)。委托常州大学药学院功能纳米材料与生物医学检测实验室完成。
具体实施方法：
正常培养NIH/3T3细胞(购自中国科学院细胞库，货号GNM6，培养、传代方法参照细胞说明书执行)。取170801、170802蛋白纯化冻干品、对照品为天然人胶原蛋白(购自Sigma，货号C7774)及牛血清白蛋白(BSA，购自生工生物(上海)股份有限公司)溶解(超纯水或1M HCl溶液)，以UV蛋白定量经验公式：C(mg/mL)＝0.144*(A215-A225)测定蛋白浓度，再以PBS(pH 7.4)稀释至0.5mg/mL。向96孔细胞培养板中加入100μL各种蛋白溶液和空白 PBS溶液对照，室温静置60min；再向每孔中加入10 5个培养状态良好的3T3细胞，37℃、5％CO 2孵育60min。以PBS清洗4次孔中细胞。使用LDH检测试剂盒(Roche，04744926001)检测OD492nm的吸光度值(具体操作参照说明书执行)。
OD492nm的吸光度可相应的表征出胶原蛋白样品的细胞粘附活性：吸光度值越高，说明蛋白粘附的细胞越多，黏附活性越高，胶原蛋白越能在短时间内帮助细胞贴壁或粘附于细胞外基质之上，更利于构建更佳的细胞外环境。结果如图19结果所示，重组人XVII型胶原蛋白170801、170802与商品化的天然人胶原蛋白相比，具有更好的细胞粘附活性。
实施例4.划痕法检测重组胶原蛋白细胞迁移活性
重组胶原蛋白的细胞迁移活性检测及分析方法参考文献Bobadilla,A.,et al.,In vitro cell migration quantification method for scratch assays.J R Soc Interface,2019.16(151):p.20180709.委托常州大学药学院功能纳米材料与生物医学检测实验室完成。
具体实施方法：
取170801、170802胶原蛋白纯化冻干品、对照品为天然人胶原蛋白(购自Sigma，货号C7774)及牛血清白蛋白(BSA，购自生工生物(上海)股份有限公司)溶解(超纯水或1M HCl溶液)，以UV蛋白定量经验公式：C(mg/mL)＝0.144*(A215-A225)测定蛋白浓度，再以DMEM无血清培养(GIBCO,货号12800017，pH 7.4)稀释至0.5mg/mL(稀释后，调节其pH稳定于7.0～7.4)。正常培养、传代NIH/3T3细胞(购自中国科学院细胞库，货号GNM6，培养、传代方法参照细胞说明书执行)。将状态良好的细胞接入6孔板，每孔按照2万细胞/mL密度接种2mL细胞悬液，培养36h。用200μL枪头制备划痕，用PBS清洗细胞3次，去除划下的细胞。于孔中加入DMEM无血清培养基稀释的蛋白溶液，继续放入37℃、5％CO 2培养箱培养，0h、24h、48h取样、拍照。使用Image J软件对细胞迁移的图片进行处理，获得初始划痕面积和无细胞空白区域面积数据，计算迁移率，迁移率＝(1-无细胞空白区域面积/初始划痕面积)*100％。
体外细胞迁移实验在一定程度上模拟了体内细胞迁移的过程，直接反映了细胞与胞外基质及基质影响下细胞之间的相互作用。细胞迁移活性是更有效表征胶原蛋白生物学活性的指标，迁移率越高，速度越快，说明胶原蛋白的生物学活性越佳。结果如图20所示，不同时间下拍摄的细胞迁移实际对比图(两黑色线内为初始及细胞迁移后划痕伤口区域)、及图21所示的计算出的细胞迁称率(Image J计算无细胞空白区域)的比较可知，重组人XVII型胶原蛋白170801、170802均有比商品化的天然人胶原蛋白更优的细胞迁移活性。
实施例5. 170801、170802重组胶原蛋白凝胶制备与兔耳瘢痕模型实验
取羧甲基纤维素钠、分别取170801、170802胶原蛋白纯化冻干品溶解于超纯水中，得到 浓度为0.01％羧甲基纤维素钠、5％胶原蛋白，将0.01％羧甲基纤维素钠和5％胶原蛋白混匀后配制为胶原蛋白凝胶，不含胶原蛋白的羧甲基纤维素钠凝胶作为阴性对照。
新西兰白化兔(中国人民解放军海军军医大学实验动物中心购进)，体重为2-3kg。新西兰白化兔在通风良好的房间内饲养，室温21℃，12小时昼夜循环，单笼饲养，在实验前饲养2周适应环境。随后进行瘢痕模型构建：实验动物接受30g/L戊巴比妥钠1.0mL/kg(30mg/kg)作耳缘静脉麻醉。确定麻醉后，以酒精擦洗消毒皮肤3遍。在兔耳内侧以直径1cm皮肤活检器划出整齐皮肤切缘，眼科剪剪出约直径1cm的全层皮肤缺损创面，暴露至耳软骨骨面。以无菌凡士林油纱覆盖创面，外面覆盖无菌纱布，包扎固定。每2-3天以碘伏对创面消毒清创，并更换创面敷料。
第21天创面愈合后分组进行瘢痕干预处理，分别使用170801胶原蛋白凝胶、170802胶原蛋白凝胶涂抹创面，配合含碘抗菌手术贴膜(Drape
Figure PCTCN2022071968-appb-000017
Minnesota Mining and Manufacturing Company，USA)、自粘性硅酮胶片敷料(
Figure PCTCN2022071968-appb-000018
Smith&Nephew，England)进行瘢痕干预处理。
使用数码照相机及皮肤镜记录1-6周瘢痕干预实验过程中的瘢痕大体，第6周予麻醉后予耳缘静脉注射过饱和氯化钾溶液处死，取耳部瘢痕样本，浸泡于固定液，予HE染色、MASSON染色，显微镜观察、拍照，具体方法可参照：[1]刘彤.重组人胶原蛋白水凝胶和人工真皮分别作为微粒皮移植覆盖物修复全层皮肤缺损的实验研究[D].中国人民解放军海军军医大学,2019.[2]李继承主编.组织学与胚胎学实验指导[M].北京：人民卫生出版社,2018.09.。
实验结果如图22所示，170801、170802胶原蛋白凝胶干预的兔耳皮肤伤口愈合后瘢痕恢复好，兔耳内侧有明显新生毛发长出，表明有明显的具有生物学功能的毛囊形成并有毛发生长，从组织切片的Masson染色、HE染色结果看，其组织中胶原蛋白纤维排列有序，已开始形成真皮组织，尤其是存在新生毛囊结构，并明确的毛发的生长(图中箭头指的圈内)，这是有效真皮组织恢复和具有生物学功能(生成毛发)的毛囊新生的明确标志；而对照组中，预后瘢痕仍为大量规则致密胶原沉积，类似于瘢痕结构，没有出现毛囊结构，无真皮组织形成，也没有任何恢复生长的兔毛出现。实验结果表明，重组人XVII型胶原蛋白170801、170802有优异组织修复和促进毛囊修复再生的生物活性。

Claims (19)

  1. 一种重组人XVII型胶原蛋白，所述胶原蛋白的氨基酸序列选自SEQ ID NO.2或SEQ ID NO.3所示的氨基酸序列。
  2. 根据权利要求1所述的胶原蛋白，其特征在于，所述胶原蛋白的肽链氨基端与羧基端设计添加蛋白质标签，所述氨基端添加Strep-Tag II标签、羧基端添加6×His Tag标签。
  3. 根据权利要求1所述的胶原蛋白，其特征在于，所述胶原蛋白具有细胞粘附活性、促细胞迁移活性、促进组织再生和促进毛囊修复再生的活性。
  4. 编码权利要求1所述胶原蛋白的多核苷酸。
  5. 根据权利要求4所述的多核苷酸，其特征在于，所述多核苷酸的核苷酸序列选自SEQ ID NO.6或SEQ ID NO.7所示的核苷酸序列。
  6. 一种重组表达载体，含有权利要求4或5所述的多核苷酸。
  7. 一种工程菌，所述工程菌含有权利要求6所述的重组表达载体。
  8. 根据权利要求7所述的工程菌，其特征在于，所述工程菌为毕赤酵母。
  9. 一种制备权利要求1所述胶原蛋白的方法，其特征在于，包括如下步骤：
    (1)工程菌的培养和筛选：将构建的工程菌经过筛选后，筛选表达量高的工程菌；
    (2)规模化高密度发酵、培养和蛋白质纯化：采用发酵罐进行发酵，对发酵上清液进行纯化，获取高纯度胶原蛋白。
  10. 根据权利要求9所述的方法，其特征在于，步骤(1)所述筛选的表达量高的工程菌为巴斯德毕赤酵母(Pichia pastoris)，保藏编号为CGMCC No.20626或CGMCC No.20627。
  11. 一种组合物，所述组合物包含权利要求1所述胶原蛋白或权利要求9所述方法制备的胶原蛋白。
  12. 一种制品，所述制品包含权利要求1所述胶原蛋白或权利要求9所述方法制备的胶原蛋白或权利要求11所述组合物，所述制品包括药物、医疗器材、生物材料、组织工程产品、化妆品或保健品。
  13. 权利要求1所述胶原蛋白、权利要求4或5所述多核苷酸、权利要求6所述重组表达载体、权利要求7所述工程菌或权利要求11所述组合物在制备制成品中的用途，所述制成品包括药物、医疗器材、生物材料、组织工程产品、化妆品 或保健品。
  14. 权利要求1所述胶原蛋白、权利要求4或5所述多核苷酸、权利要求6所述重组表达载体、权利要求7所述工程菌或权利要求11所述组合物在制备促进瘢痕愈合、组织修复或促进毛囊修复的产品中的用途。
  15. 根据权利要求14所述的用途，其特征在于，所述产品为外用制剂。
  16. 根据权利要求15所述的用途，其特征在于，所述产品为外用涂抹制剂。
  17. 根据权利要求16所述的用途，其特征在于，所述产品为外用涂抹凝胶剂。
  18. 一种细胞，所述细胞含有权利要求6所述的重组表达载体。
  19. 根据权利要求18所述的细胞，其特征在于，所述细胞为毕赤酵母。
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