Recombinant collagen functional fragment as well as production method and application thereof

Abstract

The invention provides a recombinant collagen functional fragment. The amino acid sequence of the recombinant collagen functional fragment is as shown in SEQ ID NO. 2. The invention also provides a production method and application of the recombinant collagen functional fragment. The recombinant collagen functional fragment is formed by combining alpha 1 (IV) NC1 with the effects of inhibiting angiogenesis and tumor growth and HEPV with the effects of resisting tumors and stimulating expression of collagen IV and XVIIII alpha 1 chains, shows remarkable in-vitro anti-proliferative activity on various cancer cells, and is expected to have important application in the fields of angiogenesis resistance and tumor resistance.

Description

Technical Field

[0001] The present application belongs to the field of biotechnology, and specifically relates to a recombinant collagen functional fragment and a production method and application thereof. Background Art

[0002] Type IV collagen (IV-Col) is the main component of the vascular basement membrane (VBM). After intracellular synthesis, it directly participates in the formation of the extracellular matrix as pre-collagen. VBM is an important component of blood vessels. The remodeling of VBM can provide important angiogenic and anti-angiogenic molecules to control new capillaries. The NC1 domains including endostatin and type IV collagen non-collagenous domains α1, α2, and α3 can be considered as anti-angiogenic molecules of VBM. It has been reported that α1(IV)NC1 binds to α1β1 integrin and inhibits specific integrin signaling pathways in vascular endothelial cells to induce unique effects on angiogenesis in vivo and in vitro, indicating that α1(IV)NC1 has highly specific α1β1 integrin-dependent anti-angiogenic activity and is a potential therapeutic candidate for targeting tumor angiogenesis.

[0003] FGF-, also known as βFGF, FGF, FGF-β or basic fibroblast growth factor, belongs to the heparin-binding fibroblast growth factor family. FGF- interacts with endothelial cells present on the cell surface and in the extracellular matrix through two different types of receptors, high affinity tyrosine kinase receptors (FGFR) and low affinity heparan sulfate proteoglycans (HSPG). During wound healing of normal tissues and during tumor development, FGF- acts on endothelial cells. When the VEGF pathway is blocked by anti-angiogenic drugs, FGF- expression increases, which allows tumor blood vessel formation and re-growth. The HEPV peptide is a 12kDa fragment of the collagen Vpro-α1 chain, consisting of residues Ile824-Pro950. The HEPV peptide can be used as a drug, in particular for the treatment of cancer, especially as an inhibitor of FGF-induced biological effects, more particularly as an inhibitor of FGF-induced angiogenesis processes. In fact, when administered to animals or patients, the peptide exhibits specific anti-angiogenic properties. In addition, HEPV can stimulate the expression of collagen IV and XVIII α1 chains. Summary of the invention

[0004] In view of the problems existing in the prior art, the present application provides a recombinant collagen functional fragment.

[0005] Specifically, this application involves the following aspects:

[0006] 1. A recombinant collagen functional fragment, the amino acid sequence of which is shown in SEQ ID NO.2.

[0007] 2. A nucleic acid molecule encoding the functional fragment of the protein described in item 1.

[0008] 3. The nucleic acid molecule according to item 2, wherein its sequence is shown in SEQ ID NO.1.

[0009] 4. An expression vector comprising the nucleic acid molecule described in item 2 or 3.

[0010] 5. A host cell comprising the expression vector described in item 4.

[0011] 6. The host cell according to item 5, wherein the host cell is selected from any one of Escherichia coli, Pichia pastoris, plant cells and animal cells.

[0012] 7. A method for producing a recombinant collagen functional fragment, comprising the following steps:

[0013] The host cell described in item 5 or 6 is used for expression, and then separation and purification are performed to obtain the recombinant collagen functional fragment.

[0014] 8. The method according to item 8, wherein the separation and purification method comprises ultrafiltration, ion exchange chromatography, and gel filtration chromatography.

[0015] 9. Use of the protein functional fragment described in Item 1, or the protein functional fragment encoded by the nucleic acid molecule described in Item 2 or 3, or the protein functional fragment expressed by the expression vector described in Item 4 in the preparation of anti-angiogenic and anti-tumor drugs.

[0016] 10. An anti-angiogenic and anti-tumor drug, comprising the functional protein fragment described in item 1, or the functional protein fragment encoded by the nucleic acid molecule described in item 2 or 3, or the functional protein fragment expressed by the expression vector described in item 4.

[0017] This application has the following beneficial effects:

[0018] The recombinant collagen functional fragment of the present application combines α1(IV)NC1, which has the effect of inhibiting angiogenesis and tumor growth, with HEPV, which has the effect of anti-tumor and stimulating the expression of collagen IV and XVIIIα1 chains. It shows significant in vitro anti-proliferative activity against a variety of cancer cells and is expected to have important applications in the fields of anti-angiogenesis and anti-tumor. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a diagram of the construction of the recombinant collagen expression vector plasmid;

[0020] Figure 2 This is the enzyme digestion identification diagram of the recombinant collagen expression vector;

[0021] Figure 3 This is the PCR identification diagram of the recombinant collagen positive transformant;

[0022] Figure 4 For screening high copy transformant plates using 2 g / L and 4 g / L G418;

[0023] Figure 5 This is the SDS-PAGE protein electrophoresis detection diagram of recombinant collagen. DETAILED DESCRIPTION

[0024] The present application is further described below in conjunction with examples. It should be understood that the examples are only used to further describe and illustrate the present application and are not used to limit the present application.

[0025] Unless otherwise defined, the technical and scientific terms in this specification have the same meaning as those generally understood by those skilled in the art. Although similar or identical methods and materials as described herein may be used in experiments or practical applications, materials and methods are described herein below. In the event of a conflict, the present specification including the definitions therein shall prevail. In addition, materials, methods and examples are provided for illustration only and are not restrictive. The present application is further described below in conjunction with specific embodiments, but is not intended to limit the scope of the present application.

[0026] As used herein, the terms "polypeptide," "peptide," "protein," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. That is, a description directed to a polypeptide applies equally to describing a peptide and describing a protein, and vice versa. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues is a non-naturally encoded amino acid. As used herein, the terms encompass amino acid chains of any length.

[0027] As used herein, the term "functional protein fragment" or "functional protein fragment" refers to naturally occurring or artificially synthesized proteins or polypeptides that have certain effects on the body.

[0028] As used herein, the term "nucleic acid molecule" may include those containing naturally and / or non-naturally occurring nucleotides and bases, including, for example, those with backbone modifications, and refers to polymers of nucleotides, such polymers of nucleotides may contain natural and / or non-natural nucleotides, and include, but are not limited to, DNA, RNA, and PNA. Nucleotide sequence refers to the linear sequence that constitutes a nucleic acid molecule.

[0029] In some cases, the nucleic acid molecule contains cDNA, and in some cases, the nucleic acid molecule can be modified for use in the constructs described herein, such as for codon optimization. In some cases, for the purpose of cloning into a vector, the sequence can be designed to contain a terminal restriction site sequence.

[0030] In some cases, nucleic acid molecules can be obtained from a variety of sources, such as by polymerase chain reaction (PCR) amplification of encoding nucleic acids within or isolated from one or more given cells.

[0031] As used herein, the term "polynucleotide" or "nucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides and polymers thereof in single-stranded or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides, which have binding properties similar to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specifically limited, the term also means oligonucleotide analogs, including PNA (peptide nucleic acid), DNA analogs used in antisense technology (phosphorothioate, phosphoramidate, etc.). Unless otherwise specified, a specific nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including, but not limited to, degenerate codon substitutions) and complementary sequences as well as explicitly specified sequences.

[0032] As used herein, the term "vector" is used to describe a nucleic acid molecule that can be engineered to contain a polynucleotide or multiple polynucleotides that can be cloned and amplified in a host cell. Vectors include, but are not limited to: single-stranded, double-stranded or partially double-stranded nucleic acid molecules; nucleic acid molecules containing one or more free ends, no free ends (e.g., circular); nucleic acid molecules containing DNA, RNA or both; and other polynucleotide species known in the art. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA fragments can be inserted, such as by standard molecular cloning techniques. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors and episomal mammalian vectors having a bacterial origin of replication). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell after introduction into the host cell, thereby replicating with the host genome. In addition, certain vectors are capable of directing the expression of those genes to which they are operably linked. Such vectors are referred to herein as "expression vectors." The recombinant expression vector may contain the nucleic acid of the present application in a form suitable for expressing the nucleic acid in a host cell, which means that the recombinant expression vector includes one or more regulatory elements, which can be selected based on the host cell used for expression and can be operably linked to the nucleic acid sequence to be expressed.

[0033] As used herein, the term "expression" includes any step involved in the production of the variant including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0034] As used herein, the term "expression vector" means a linear or circular DNA molecule comprising a polynucleotide encoding a variant and operably linked to other nucleotides that provide for its expression.

[0035] As used herein, the term "host cell" means any cell type that is susceptible to transformation, transfection, transduction, etc. of a nucleic acid construct or expression vector comprising the polynucleotides of the present application. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The host cell can be any cell useful in the production of recombinant collagen in the present application.

[0036] As used herein, the term "recombinant collagen functional fragment" refers to a collagen functional fragment produced by recombinant technology, wherein the DNA or RNA encoding the expressed protein is usually inserted into a suitable expression vector, which is then used to transform a host cell to produce the protein. In some exemplary embodiments, the DNA or RNA encoding the expressed peptide, polypeptide or protein is inserted into a host chromosome by homologous recombination or other means known in the art, and is therefore used to transform a host cell to produce the protein.

[0037] As described above, the type IV collagen non-collagenous domain 1 [α1(IV)NC1] is derived from the carboxyl terminus of type IV collagen, which inhibits angiogenesis and tumor growth in vivo and is identified as an endogenous anti-angiogenesis and anti-tumor factor, and also has anti-lymphangiogenesis effects. The amino acid sequence of the type IV collagen non-collagenous domain 1 [α1(IV)NC1] is shown in SEQ ID NO.3:

[0038] IKGDRGEIGPPPGPRGEDGPEGPKGRGGPNGDPGPLGPPGEKGKLGVPGL PGYRGRQGPKGSIGFPGFPGANGEKGGRGTPGKPGPRGQRGPTGPRGERGP RGITGKPGPKGNSGGDGPAGPPGERGPVSIGYLLVKHSQTDQEP(SEQ ID NO.3)

[0039] HEPV peptide is a 12 kDa fragment of collagen Vpro-α1 chain, consisting of residues Ile824-Pro950. Studies have shown that Hepv peptide can be used as a drug, especially for the treatment of cancer, especially as an inhibitor of FGF-induced biological effects, more especially as an inhibitor of FGF-induced angiogenesis. And there are data showing that HEPV can stimulate the expression of collagen IV and XVIII α1 chain. The amino acid sequence of HEPV is shown in SEQ ID NO.4:

[0040] VSIGYLLVKHSQTDQEPMCPVGMNKLWSGYSLLYFEGQEKAHNQDLGLAGSCLARFSTMPFLYCNPGDVCYYASRNDKSYWLSTTAPLPPMVAEDEIKPYISRCSVCEAPAIAI AVHSQDVSIPHCPAGWRSLWIGYSFLMHTAAGDEGGGQSLVSPGSCLEDFRATPFIECNGGRGTCHYYANKYSFWLTTIPEQSFQGSPSADTLKAGLIRTHISRCQVCMKNL(SEQ ID NO.4).

[0041] The present application provides a recombinant collagen functional fragment, which is obtained by combining α1(IV)NC1 with the effects of inhibiting angiogenesis and tumor growth with HEPV with the effects of anti-tumor and stimulating the expression of collagen IV and XVIIIα1 chains, and its amino acid sequence is shown in SEQ ID NO.2:

[0042] IKGDRGEIGPPGPRGEDGPEGPKGRGGPNGDPGPLGPPGEKGKLGVPGLPGYRGRQGPKGSIGFPGFPGANGEKGGRGTPGKPGPRGQRGPTGPRGERGPRGITGKPGPKGNSGGDGPAGPPGERGPVSIGYLLVKHSQTDQEPVSIGYLLVKHSQTDQEPMCPVGMNKLWSGYSLLYFEGQEKAHN QDLGLAGSCLARFSTMPFLYCNPGDVCYYASRNDKSYWLSTTAPLPMMPVAEDEIKPYISRCSVCEAPAIAIAVHSQDVSIPHCPAGWRSLWIGYSFLMHTAAGDEGGGQSLVSPGSCLEDFRATPFIECNGGRGTCHYYANKYSFWLTTIPEQSFQGSPSADTLKAGLIRTHISRCQVCMKNL(SEQ ID NO.2).

[0043] The recombinant collagen functional fragment of the present application is obtained by combining α1(IV)NC1, which has the effect of inhibiting angiogenesis and tumor growth, with HEPV, which has the effect of anti-tumor and stimulating the expression of collagen IV and XVIIIα1 chains. Therefore, it has the functions of both α1(IV)NC1 and HEPV, and shows significant in vitro anti-proliferative activity against various cancer cells such as breast cancer, ovarian cancer, breast ductal carcinoma, and liver cancer. It is expected to have important applications in the fields of anti-angiogenesis and anti-tumor.

[0044] The present application also provides a nucleic acid molecule, which encodes the above-mentioned recombinant collagen functional fragment.

[0045] In a specific embodiment, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO: 1:

[0046] ATTAAAGGAGATCGAGGTGAAATTGGTCCTCCCGGACCAAGAGGTGAAGACGGTCCAGA

[0047] AGGTCCAAAAGGTAGAGGAGGTCCCAACGGTGACCCCGGTCCACTTGGACCTCCAGGA

[0048] GAAAAGGGTAAACTAGGTGTTCCTGGTCTCCAGGATACAGAGGTCGTCAAGGACCAAA

[0049] AGGTTCTATTGGTTTCCCCGGTTTTCCTGGAGCAAATGGAGAGAAAGGTGGTAGAGGTAC

[0050] ACCTGGAAAACCTGGCCCAAGGGGACAGAGAGGACCTACTGGACCCAGGGGTGAGAGA

[0051] GGACCTAGAGGTATTACTGGTAAACCTGGTCCTAAGGGCAATAGTGGTGGAGATGGTCCA

[0052] GCTGGTCACCAGGAGAGAGAGGCCCAGTTTCTATTGGATATCTGTTGGTCAAACATTCT

[0053] CAAACTGATCAAGAGCCTGTCAGTATTGGCTACCTTTTGGTCAAACATTCACAAACCGAC

[0054] CAGGAACCTATGTGCCCTGTTGGTATGAACAAGCTGTGGTCCGGTTACTCTTTGCTTTACT

[0055] TTGAGGGTCAAGAAAAGGCTCACAATCAGGATTTGGGTTTGGCAGGTTCCTGCTTGGCA

[0056] AGATTCTCTACTATGCCTTTTCTTTACTGCAATCCTGGCGACGTTTGCTACTACGCTTCTAG

[0057] GAACGACAAAAGTTACTGGCTAAGTACCACAGCTCCTCTGCCCATGATGCCAGTCGCTGA

[0058] GGACGAGATTAAACCATACATCTCTAGATGTTCTGTTTGCGAAGCTCCAGCTATCGCTATT

[0059] GCTGTGCACTCCCAGGATGTTTCCATTCCTCACTGCCCTGCAGGATGGAGGAGTTTGTGG

[0060] ATCGGTTACTCATTCTTGATGCACACAGCCGCAGGTGATGAGGGTGGAGGACAAAGTTTG

[0061] GTTTCACCAGGATCCTGTCTAGAGGATTTTAGAGCCACCCCTTTTATTGAATGTAATGGCG

[0062] GTAGAGGTACTTGTCACTACTATGCCAATAAGTATTCTTTTTGGTTGACAACCATTCCTGA

[0063] GCAGTCCTTCCAGGGTTCACCTTCTGCAGATACTTTGAAAGCCGGTTTAATTAGAACTCACATTTCCCGATGCCAAGTTTGTATGAAAAACCTT(SEQ ID NO.1)

[0064] This application provides an expression vector, which contains the above nucleic acid molecule.

[0065] For example, one or more nucleic acids encoding the above-mentioned recombinant collagen functional fragments are cloned into one or more suitable expression vectors, which can be any suitable recombinant expression vector and can be used to transform or transfect any suitable host. Suitable vectors include those designed for propagation and amplification or for expression or for both, such as plasmids and viruses.

[0066] The vector may contain regulatory sequences (such as transcriptional and translational initiation and termination codons) that are specific for the type of host (e.g., bacteria, fungi, plants, or animals) into which the vector is to be introduced, and whether the vector is DNA-based or RNA-based, as appropriate.

[0067] In a specific embodiment, the expression vector is pPIC9, pPIC3K, pPIC9k, pPIczA or pPIcza, etc.

[0068] The present application provides a host cell, which comprises the above-mentioned expression vector.

[0069] In order to produce recombinant collagen functional fragments, nucleic acid encoding recombinant collagen functional fragments can be isolated and inserted into one or more vectors to further clone and / or express in host cells. Conventional techniques (e.g., by using oligonucleotide probes that can specifically bind to genes encoding recombinant collagen) can be used to easily separate and sequence such nucleic acids.

[0070] 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.

[0071] Methods for introducing vectors into host cells are well known, for example, using electroporation to introduce vectors into host cells.

[0072] In a specific embodiment, the host cell is selected from any one of Escherichia coli, Pichia pastoris, plant cells and animal cells.

[0073] In a specific embodiment, the host cell is Pichia pastoris.

[0074] This application uses Pichia pastoris as an exogenous expression host. Among them, the yeast expression system has the advantages of fast growth and simple operation of prokaryotes, and the advantage of eukaryotic post-translational modification of proteins, and can express complex structural proteins such as glycoproteins. The yeast expression system mainly includes the Saccharomyces cerevisiae expression system and the Pichia pastoris expression system. Saccharomyces cerevisiae has certain limitations, such as low yield, easy loss of expression plasmids, excessive glycosylation of exogenous genes, poor secretion effect, and unsuitability for high-density fermentation.

[0075] The present application provides a method for producing a recombinant collagen functional fragment, which comprises the following steps:

[0076] The above-mentioned host cells are used for expression and then separated and purified to obtain the product.

[0077] 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.

[0078] The present application does not impose any limitation on the expression mode, which can be confirmed as needed. For example, the expression is induced expression. For induced expression, the inducer can be IPTG, β-galactoside, methanol, ethanol, etc., preferably methanol.

[0079] In a specific embodiment, the specific culture conditions are as follows: randomly pick a single clone from the MD plate and place it in YPD medium, and culture it overnight at 28°C and 200 r / min; inoculate 5%-8% (v / v) of the bacterial solution into liquid BMGY medium, and culture it at 28°C and 200 r / min. When the bacterial density grows to OD 5-15, collect the bacteria by centrifugation at 4000 r / min for 5 min, resuspend the precipitate with liquid BMMY medium, and then inoculate it into BMMY medium (50 mL / 500 mL Erlenmeyer flask, pH 6.0) to make its OD about 2.0, culture it at 28°C and 220 r / min, and add 0.5% methanol (v / v) every day.

[0080] 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, ion exchange chromatography, and gel filtration chromatography can be used.

[0081] The present application also provides the use of the above-mentioned recombinant collagen functional fragment, the recombinant collagen functional fragment encoded by the above-mentioned nucleic acid molecule, or the recombinant collagen functional fragment expressed by the above-mentioned expression vector in the preparation of anti-angiogenesis and anti-tumor drugs.

[0082] In a specific embodiment, the tumor is breast cancer, ovarian cancer, breast ductal carcinoma, liver cancer or gastric cancer.

[0083] The present application also provides an anti-angiogenesis and anti-tumor drug, which includes the above-mentioned recombinant collagen functional fragment, the recombinant collagen functional fragment encoded by the above-mentioned nucleic acid molecule, or the recombinant collagen functional fragment expressed by the above-mentioned expression vector.

[0084] In a specific embodiment, the tumor is breast cancer, ovarian cancer, breast ductal carcinoma, liver cancer or gastric cancer.

[0085] Example

[0086] Example 1

[0087] Synthesis of gene sequence of recombinant collagen functional fragment and construction of expression vector

[0088] The nucleotide sequence of synthetic recombinant collagen (nucleotide sequence is shown in SEQ ID NO. 1) has a NotI restriction site introduced at its 5' end and an EcoRI restriction site introduced at its 3' end.

[0089] The synthetic gene and pPIC9 expression vector were double-digested with restriction endonucleases NotI and EcoRI, the target fragment was purified by gel recovery, and then the digested fragments were connected with T4 DNA ligase, connected at 4°C overnight, transformed into Escherichia coli DH5α competent cells, positive clones were screened by LB plates, single clones were randomly picked, recombinant plasmids were extracted, and positive clones were identified by restriction digestion. The recombinant plasmid was then double-digested with NotI and EcoRI, the target fragment was recovered by gel recovery, and then ligated to the pPIC9K vector fragment digested with the same enzymes to obtain the recombinant plasmid pPIC9K-col-p for expression, and was identified as a positive clone by double digestion with BamHI and NCoI, that is, the target gene was successfully inserted into the expression vector. The construction process of the recombinant plasmid pPIC9K-col-p is as follows: Figure 1 As shown, the results of double digestion with BamHI and NCoI are shown in Figure 2 shown.

[0090] Example 2 Pichia pastoris strain transformation and plate screening

[0091] The recombinant plasmid pPIC9K-col-p and the blank plasmid pPIC9K were digested with restriction endonuclease SalI, and 5ug-20ug of plasmid was electroporated into Pichia pastoris GS115 competent cells at a voltage of 1.5kV, a capacitance of 25uF, a resistance of 200Ω, and a discharge time of 4.0ms. 1.0mL of ice-cold 1.0mol / L sorbitol was immediately added and the MD plate (His - ), cultured at 28-30°C, and screened for His +Transformants. Hundreds of transformants were randomly selected, and colony PCR was performed using the original plasmid as a control to select positive transformants (such as Figure 3 As shown in the figure, according to the designed primers, the PCR result shows a band at 450 bp, which is a positive transformant); Figure 4 As shown, G418 was used to screen the positive transformants for high copy number, and the concentrations of G418 were 2 g / L and 4 g / L, respectively, in order to screen the transformants with high copy number.

[0092] Example 3 Fermentation screening of recombinant Pichia pastoris strains

[0093] Positive strains were randomly selected (blank plasmid transformed strains were selected as controls) for preliminary screening by shake flask fermentation. The screening method was as follows: single clones were randomly selected from MD plates and placed in YPD medium, and cultured overnight at 28°C, 200 r / min; 5%-8% (v / v) bacterial solution was added to liquid BMGY medium, and cultured at 28°C, 200 r / min. When the bacterial density grew to OD 5-15, the bacterial cells were collected by centrifugation at 4000 r / min for 5 min, and the precipitate was resuspended in liquid BMMY medium, and then inoculated into BMMY medium (50 mL / 500 mL Erlenmeyer flask, pH 6.0) to make its OD about 2.0, and cultured at 28°C, 220 r / min, with 0.5% methanol (v / v) added every day. During this period, samples were taken every 12 hours to detect enzyme activity, pH, biomass, and protein expression levels to be detected, and protein expression levels were detected by SDS-PAGE after 48 hours to screen out strains with higher expression levels.

[0094] Example 4 Isolation and purification of target protein from the supernatant of expression of recombinant Pichia pastoris strain

[0095] First, the ultrafilter and the large-pore (10KDa) ultrafiltration membrane were balanced with a phosphate buffer solution with a pH of 6.0 and a concentration of 20 mmol / L, and the inlet and outlet pressures were maintained at 1.3 bar and 0.3 bar, respectively. Then, the supernatant of the expression of the recombinant strain of Pichia pastoris was concentrated. When it was concentrated to about 1 L, it was diluted with 2 times the volume of the above phosphate buffer, and then ultrafiltration was continued to about 1 L. This was diluted 3 times, and the concentrated solution was collected for standby use. The chromatography column was balanced with a phosphate balance buffer with a concentration of 20 mmol / L and a pH of 6.0 for 2 to 3 column volumes, and the above ultrafiltered concentrated solution was loaded onto the column for cation exchange chromatography.

[0096] The specific steps are: balance the chromatography column with a 20mmol / L, pH 6.0 phosphate balance buffer for 3 column volumes, wash with a 20mmol / L, pH 6.8 phosphate buffer for 2 column volumes, and then use a 20mmol / L, pH 6.8 phosphate buffer containing 0.5M NaCl to linearly elute from 0-0.5M NaCl for 10 column volumes after the baseline is stable, and collect the eluate. The eluate sample obtained by cation exchange chromatography is ultrafiltered and concentrated to a certain volume through an ultrafiltration tube with a molecular weight cutoff of 5KDa, with the protein concentration not exceeding 50mg / ml. A 1.6 cm x 80 cm Sephadex G-75 chromatography column was used for gel filtration chromatography. Two column volumes were first balanced with 20 mM Tris-HCl, pH 7.4, containing 0.15 M NaCl buffer. The concentrated sample separated by cation exchange chromatography was then loaded and eluted at a flow rate of 0.5 ml / min. The collected solution was detected at a wavelength of 280 nm. The eluate curve had only a single absorption peak. The peak eluate was collected and vacuum freeze-dried in a conventional manner to obtain a solid protein powder.

[0097] Example 5 Identification and analysis of protein purity of recombinant collagen functional fragments

[0098] The protein samples obtained above were subjected to SDS-PAGE electrophoresis analysis and Coomassie Brilliant Blue staining to detect protein purity. The results are as follows Figure 5 shown.

[0099] The purified protein sample was sent to Beijing Biotech Biotechnology Co., Ltd. for full sequence mass spectrometry identification, and the sequence was shown to be:

[0100] IKGDRGEIGPPGPRGEDGPEGPKGRGGPNGDPGPLGPPGEKGKLGVPGLPGYRGRQGPKGSIGFPGFPGANGEKGGRGTPGKPGPRGQRGPTGPRGERGPRGITGKPGPKGNSGGDGPAGPPGERGPVSIGYLLVKHSQTDQEPVSIGYLLVKHSQTDQEPMCPVGMNKLWSGYSLLYFEGQEKAHN QDLGLAGSCLARFSTMPFLYCNPGDVCYYASRNDKSYWLSTTAPLPMMPVAEDEIKPYISRCSVCEAPAIAIAVHSQDVSIPHCPAGWRSLWIGYSFLMHTAAGDEGGGQSLVSPGSCLEDFRATPFIECNGGRGTCHYYANKYSFWLTTIPEQSFQGSPSADTLKAGLIRTHISRCQVCMKNL(SEQ ID NO.2).

[0101] Example 6 Detection of anti-tumor proliferation activity of recombinant collagen functional fragments

[0102] Select cells in good condition, among which four different tumor cells, MCF-7 (human breast cancer cells), SKOV-3 (human ovarian cancer cells), T47D (human breast ductal cancer cells), Hepg-2 (human liver cancer cells), and GES-1 (human gastric mucosal epithelial cells) normal cells were used as controls, and Fulvestrant was selected as a positive control. Each group of duplicate wells was 6. 100 μL per well was added with 1×10 4 The cells were inoculated into a 96-well plate and cultured in a saturated humidity, 37°C, 5% CO2 incubator for 24 hours. The experimental group was treated with 100 μL of 1 mg / ml recombinant collagen solution, and the blank control group was treated with an equal amount of normal cell culture fluid for 48 hours. 10 μL of MTT reagent (5 mg / ml) was added to each well and incubated for 4 hours. After adding 100 μL of DMSO, the absorbance at 570 nm was detected by an enzyme marker. The inhibition rate was calculated based on the absorbance, and the maximum half inhibitory concentration (IC50) value was calculated by software simulation.

[0103] Among them, inhibition rate (%) = (OD value of control group - OD value of experimental group) / OD value of control group

[0104] The results of the IC50 tests are shown in Table 1.

[0105] Table 1 IC50 values ​​of recombinant collagen against four tumor cells

[0106]

[0107] The results showed that the recombinant collagen of the present application exhibited significant in vitro anti-proliferative activity against most cancer cells, while causing less damage to normal cells.

Claims

1. A recombinant collagen functional fragment, the amino acid sequence of which is shown in SEQ ID NO.

2.

2. A nucleic acid molecule encoding the functional fragment of the protein according to claim 1.

3. The nucleic acid molecule according to claim 2, wherein Its sequence is shown in SEQ ID NO.

1. An expression vector comprising the nucleic acid molecule according to claim 2 or 3. A host cell comprising the expression vector according to claim 4 .

6. The host cell according to claim 5, wherein the host cell is selected from any one of Escherichia coli, Pichia pastoris, plant cells and animal cells.

7. A method for producing a recombinant collagen functional fragment, comprising the following steps: The host cell according to claim 5 or 6 is used for expression, and then separation and purification are performed to obtain the recombinant collagen functional fragment.

8. The method of claim 8, wherein the separation and purification method comprises ultrafiltration, ion exchange chromatography, and gel filtration chromatography.

9. Use of the functional protein fragment according to claim 1, or the functional protein fragment encoded by the nucleic acid molecule according to claim 2 or 3, or the functional protein fragment expressed by the expression vector according to claim 4 in the preparation of anti-angiogenesis and anti-tumor drugs.

10. An anti-angiogenesis and anti-tumor drug, comprising the functional protein fragment of claim 1, or the functional protein fragment encoded by the nucleic acid molecule of claim 2 or 3, or the functional protein fragment expressed by the expression vector of claim 4.

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