CN119241689A

CN119241689A - Recombinant hydroxylated human type III collagen with cell adhesion, preparation method and application thereof

Info

Publication number: CN119241689A
Application number: CN202411349697.3A
Authority: CN
Inventors: 李伟娜; 张雅倩; 范代娣; 袁艳婷; 邓鑫月; 江奇逸
Original assignee: NORTHWEST UNIVERSITY
Current assignee: NORTHWEST UNIVERSITY
Priority date: 2024-09-26
Filing date: 2024-09-26
Publication date: 2025-01-03

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to recombinant hydroxylated human III-type collagen, and a preparation method and application thereof. A recombinant hydroxylated human III type collagen with cell adhesion has an amino acid sequence shown in SEQ ID NO. 1. By replacing 9 different sites of the full-length natural III-type collagen, 4 natural integrin sites with lower adhesiveness are replaced under the condition of not changing the total length of the natural full-length amino acid, and meanwhile, the terminal peptide is reserved, so that the three-helix structure has the potential. The adhesiveness of the collagen is improved by more than 27% by the modification mode, the molecular weight of the collagen is moderate, the collagen is easy to prepare, and the collagen is stably expressed by hydroxylation.

Description

Recombinant hydroxylated human III type collagen with cell adhesion, preparation method and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to recombinant hydroxylated human type III collagen with cell adhesion, and a preparation method and application thereof.

Background

Collagen is one of the most important and abundant proteins in mammals, being structural proteins found in human skin, connective tissue and bone, and other tissues. The content of collagen in the human body is about 30% of the total protein. Collagen is a structural protein, which is the main component of the extracellular matrix. Type III collagen is twisted into a triple helix by three peptide chains which are curled rightward. Primary structure analysis shows that the segment sequence with long polypeptide chain is repeated by Gly-X-Y amino acid sequence. Where X is typically proline and Y is typically hydroxyproline and hydroxylysine, the latter two amino acids being rare in other proteins.

The natural collagen sequence has poor adhesion due to the presence of 4 natural integrin sites with low adhesion.

Disclosure of Invention

The invention aims to provide a recombinant hydroxylated human III type collagen with cell adhesion, a preparation method and application thereof, and the adhesion of the collagen is improved.

The invention adopts the technical scheme that the recombinant human type III collagen with cell adhesion has an amino acid sequence shown as SEQ ID NO.1 and a nucleotide sequence shown as SEQ ID NO. 2.

A sequence with hydroxylation function and enhanced adhesiveness of protein coded by SEQ ID NO.1 has an amino acid sequence shown as SEQ ID NO.4 and a nucleotide sequence shown as SEQ ID NO. 5.

The recombinant human type III collagen is used for preparing a cell adhesion promoter.

An expression vector comprising the nucleotide sequence set forth in SEQ ID No. 2.

An expression vector comprising the nucleotide sequence set forth in SEQ ID No. 5.

A host cell comprising the expression vector of any one of the above.

The host cell is any one of pichia pastoris, saccharomyces cerevisiae, escherichia coli and bacillus subtilis.

A method for preparing recombinant human type III collagen by using the host cell comprises culturing the host cell in a culture medium, inducing the expression of the recombinant human type III collagen, and purifying to obtain the recombinant human type III collagen.

The purification method is selected from any one of salting out method, ultrafiltration method, affinity chromatography method and gel filtration chromatography method.

Compared with the prior art, the invention has the beneficial effects that by replacing 9 different sites of the full-length natural III-type collagen, 4 natural integrin sites with lower adhesiveness are replaced under the condition of not changing the total length of the natural full-length amino acid, and meanwhile, the terminal peptide is reserved, so that the terminal peptide has the potential of forming a triple helix structure. The modified collagen has the advantages of improved adhesion by more than 27%, moderate molecular weight, easy preparation, and stable expression due to hydroxylation

The recombinant collagen of the invention does not remove the full-length chain sequences of the C-end and the N-end of the collagen coding region, and has the capability of forming a triple-helical structure by combining hydroxylation.

The recombinant collagen prepared by the invention is expressed by pichia pastoris engineering bacteria, the protein has no endotoxin hidden trouble, the protein does not carry histidine tag, and the protein can be purified by a molecular sieve, so that the target protein can be obtained directly, and extra histidine tag sequence cutting is not needed.

The recombinant collagen prepared by the method can effectively perform immune response, inflammatory response, coagulation, tumor metastasis and wound healing.

The preparation method of the recombinant human hydroxylated III-type collagen is suitable for industrialized mass production, and the prepared product has no animal-derived infection source, so that the biological safety is higher.

Drawings

FIG. 1 shows the enzyme digestion electrophoresis pattern of the target gene plasmid, A shows the enzyme digestion electrophoresis pattern of the target gene plasmid, and B shows the enzyme digestion electrophoresis pattern of the hydroxylation gene plasmid.

FIG. 2 shows colony PCR of plasmid transformants containing the target gene.

FIG. 3 shows colony PCR of plasmid transformants containing hydroxylation gene.

Fig. 4 is a graph showing the result of crystal violet staining, and BSA, type III native collagen, type III recombinant collagen, and type III recombinant hydroxylated collagen are sequentially shown from left to right.

FIG. 5 is a graph of the results of AO/EB staining, with BSA, native collagen type III, recombinant collagen type III, and recombinant hydroxylated collagen type III in that order from left to right.

Detailed Description

In order that those skilled in the art will better understand the technical scheme of the present invention, the present invention will be further described with reference to specific embodiments and drawings.

In the description of the present invention, unless otherwise specified, all reagents are commercially available and methods are conventional in the art.

The invention concept of the invention is that by replacing 9 different sites of the full-length natural III type collagen, the function of the full-length natural III type collagen is enhanced under the condition of not changing the total length of natural full-length amino acids, 4 natural integrin sites with lower adhesiveness are replaced, and meanwhile, the terminal peptide is reserved to have the potential of forming a triple helix structure. Hydroxyproline plays an important role in maintaining the triple helix structure and biological properties of collagen during synthesis. Mammalian cells having prolyl 4-hydroxylase can directly produce hydroxylated collagen by post-translational modification.

The invention aims to provide a recombinant hydroxylated collagen with cell adhesion, which can effectively support the cell adhesion and is an important molecular basis for a series of important physiological and pathological processes such as immune response, inflammatory response, coagulation, tumor metastasis, wound healing and the like. Can be widely applied to the fields of foods, health products, biological medicines and the like.

The invention provides a III type recombinant collagen with cell adhesion, and the amino acid sequence of the III type recombinant collagen is shown as SEQ ID NO. 1.

SEQ ID NO.1:

MMSFVQKGSWLLLALLHPTIILAQQEAVEGGCSHLGQSYADRDVWKPEPCQICVCDSGSVLCDDIICDDQELDCPNPEIPFGECCAVCPQPPTAPTRPPNGQGPQGPKGDPGPPGIPGRNGDPGIPGQPGSPGSPGPPGICESCPTGPQNYSPQYDSYDVKSGVAVGGLAGYPGPAGPPGPPGPPGTSGHPGSPGSPGYQGPPGEPGQAGPSGPPGPPGAIGPSGPAGKDGESGERGFPGERGVQGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGERGFPGERGFPGERGVQGPMGERGFPGERGVQGLPGAAGARGNDGARGSDGQPGPPGPPGTAGFPGSPGAKGEVGPAGSPGSNGAPGQRGEPGPQGHAGAQGPPGPPGINGSPGGKGEMGPAGIPGAPGLMGARGPPGPAGANGAPGLRGGAGEPGKNGAKGEPGPRGERGEAGIPGVPGAKGEDGKDGSPGEPGANGERGFPGERGVQGFRGPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGERGFPGERGVQGLAGAPGLRGGAGPPGPEGGKGAAGPPGPPGAAGTPGERGFPGERGVQGSPGPKGDKGEPGGPGADGVPGKDGPRGPTGPIGPPGPAGQPGDKGEGGAPGLPGIAGPRGSPGERGETGPPGPAGFPGAPGQNGERGFPGERGVQGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGERGSPGGPGAAGFPGARGLPGPPGSNGNPGPPGPSGSPGKDGPPGPAGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGPLGIAGITGARGLAGPPGMPGPRGSPGPQGVKGESGKPGANGLSGERGPPGPQGLPGLAGTAGEPGRDGNPGSDGLPGRDGSPGGKGDRGENGSPGAPGAPGHPGPPGPVGERGFPGERGVQGPAGPAGAPGPAGSRGAPGPQGPRGDKGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGAIGSPGPAGPRGPVGPSGPPGKDGTSGHPGPIGPPGPRGNRGERGSEGSPGHPGQPGPPGPPGAPGPCCGGVGAAAIAGIGGEKAGGFAPYYGDEPMDFKINTDEIMTSLKSVNGQIESLISPDGSRKNPARNCRDLKFCHPELKSGEYWVDPNQGCKLDAIKVFCNMETGETCISANPLNVPRKHWWTDSSAEKKHVWFGESMDGGFQFSYGNPELPEDVLDVHLAFLRLLSSRASQNITYHCKNSIAYMDQASGNVKKALKLMGSNEGEFKAEGNSKFTYTVLEDGCTKHTGEWSKTVFEYRTRKAVRLPIVDIAPYDIGGPDQEFGVDVGPVCFL.

The invention provides a III type recombinant collagen with cell adhesion, which is used for optimizing codons aiming at host cell expression, and signal peptide cleavage sites and EcoR I and Not I cleavage sites are respectively added at two ends in the design process so as to be beneficial to later gene operation. Through the optimization, the nucleotide sequence is shown as SEQ ID NO. 2.

SEQ ID NO.2:

ATGATGTCTTTTGTTCAGAAAGGTTCCTGGTTGTTGTTGGCCTTGTTACATCCAACTATTATTTTGGCTCAGCAAGAAGCTGTTGAAGGAGGTTGTTCTCACTTGGGTCAAAGTTATGCTGATAGAGATGTTTGGAAACCTGAACCTTGTCAAATTTGCGTTTGTGATAGTGGTTCTGTTCTTTGTGATGATATTATTTGTGATGATCAGGAATTGGACTGTCCTAACCCTGAGATCCCATTTGGTGAATGTTGTGCAGTTTGTCCACAACCACCTACAGCACCAACTAGACCACCTAACGGTCAAGGTCCACAAGGTCCTAAAGGTGACCCTGGACCTCCTGGTATCCCAGGTAGAAATGGTGACCCAGGTATTCCAGGTCAACCAGGATCTCCTGGTAGTCCTGGTCCACCTGGTATTTGTGAGTCTTGTCCAACTGGTCCACAGAATTACTCTCCACAATATGACTCCTATGATGTTAAATCTGGAGTTGCTGTTGGTGGATTAGCCGGTTATCCAGGTCCTGCTGGTCCTCCCGGTCCACCTGGACCACCTGGTACTTCTGGTCATCCAGGTTCTCCTGGTTCTCCAGGTTATCAAGGACCTCCAGGTGAACCAGGTCAAGCTGGTCCATCTGGACCCCCAGGTCCACCTGGTGCTATAGGTCCTTCAGGACCAGCAGGAAAAGATGGTGAATCTGGTGAAAGAGGTTTTCCAGGTGAAAGGGGTGTTCAAGGACCTCCAGGTATTAAGGGTCCAGCTGGAATCCCTGGTTTCCCAGGTATGAAGGGTCATAGAGGTTTTGATGGTAGAAATGGTGAAAAAGGTGAAACAGGTGAGAGAGGTTTTCCTGGTGAGAGAGGATTCCCAGGTGAACGAGGTGTTCAAGGTCCAATGGGTGAACGTGGTTTCCCAGGTGAAAGAGGCGTTCAAGGACTACCTGGTGCTGCTGGTGCTAGAGGTAATGATGGAGCTAGAGGTTCAGATGGTCAACCAGGTCCTCCTGGTCCACCAGGTACAGCTGGATTTCCTGGATCTCCAGGTGCTAAGGGTGAAGTTGGTCCAGCTGGTTCTCCAGGATCTAATGGAGCTCCAGGACAAAGAGGTGAACCTGGACCACAAGGTCATGCTGGAGCTCAAGGACCACCTGGTCCTCCAGGAATTAATGGTTCTCCTGGTGGCAAGGGTGAAATGGGACCTGCTGGAATCCCAGGTGCTCCAGGATTAATGGGTGCTAGAGGTCCTCCTGGACCTGCTGGAGCTAATGGAGCTCCTGGTTTGAGAGGTGGAGCTGGTGAGCCAGGTAAAAATGGTGCTAAAGGTGAACCTGGTCCAAGAGGAGAAAGAGGTGAAGCTGGTATTCCAGGAGTTCCAGGAGCTAAAGGTGAAGATGGTAAAGATGGATCACCAGGAGAACCAGGTGCTAACGGTGAAAGAGGATTTCCCGGTGAACGTGGAGTTCAAGGATTTCGTGGACCAGCTGGACCTAATGGTATTCCTGGTGAAAAAGGACCAGCTGGAGAAAGAGGAGCTCCAGGTCCAGCAGGTCCTAGAGGTGCTGCAGGTGAACCAGGTAGAGATGGTGTTCCAGGAGGACCAGGTATGAGAGGTATGCCAGGAAGTCCAGGTGGACCAGGTTCTGACGGAAAGCCAGGTCCTCCAGGTTCACAAGGTGAATCTGGTAGACCAGGTCCTCCAGGTCCATCTGGACCAAGAGGTCAACCTGGTGTTATGGGATTTCCAGGACCTAAAGGTAATGATGGTGCTCCAGGTAAAAATGGTGAAAGAGGTGGTCCTGGTGGTCCAGGACCACAAGGACCACCAGGAAAAAATGGTGAAACTGGACCACAAGGTCCACCAGGACCTACTGGACCAGGAGGAGATAAAGGTGACACTGGACCACCAGGACCACAAGGATTGCAAGGACTTCCTGGTACTGGTGGACCACCAGGTGAAAATGGTAAACCTGGTGAACCAGGACCAAAAGGTGACGCAGGAGCACCAGGTGCACCAGGAGGTAAAGGAGAACGTGGATTTCCAGGTGAAAGAGGCGTTCAAGGATTAGCTGGTGCACCAGGATTGAGAGGTGGTGCAGGACCACCAGGTCCTGAAGGAGGTAAAGGAGCTGCTGGTCCTCCTGGACCTCCAGGAGCTGCTGGTACTCCTGGTGAGAGAGGATTTCCTGGTGAAAGGGGAGTTCAAGGTAGTCCAGGTCCTAAAGGTGACAAGGGAGAACCTGGAGGTCCAGGTGCTGATGGTGTTCCAGGTAAAGATGGTCCAAGAGGTCCAACTGGTCCTATTGGTCCACCAGGACCTGCTGGTCAACCTGGAGATAAAGGTGAAGGTGGTGCTCCAGGTTTGCCAGGAATTGCTGGTCCTAGAGGTTCTCCAGGTGAGAGAGGTGAAACAGGTCCACCAGGTCCTGCTGGTTTTCCAGGTGCACCAGGTCAAAACGGTGAAAGAGGATTTCCTGGTGAACGTGGTGTTCAAGGTGAAAAAGGTGAAGGTGGCCCACCAGGTGTTGCTGGTCCACCTGGAGGATCTGGTCCAGCTGGTCCTCCAGGTCCACAAGGTGTTAAGGGTGAAAGAGGTTCCCCAGGTGGTCCAGGTGCTGCTGGTTTCCCTGGTGCTAGAGGTTTGCCAGGACCACCAGGTTCTAACGGTAACCCTGGTCCACCTGGTCCATCCGGTTCCCCTGGTAAAGATGGTCCACCAGGTCCTGCTGGTAATACTGGTGCTCCTGGTAGTCCAGGTGTTTCCGGTCCAAAGGGAGATGCTGGTCAACCAGGTGAAAAGGGTTCCCCAGGTGCTCAAGGTCCTCCAGGTGCTCCAGGTCCATTAGGAATTGCTGGTATTACTGGTGCTAGAGGTTTGGCTGGTCCACCTGGAATGCCAGGACCAAGAGGTTCTCCAGGTCCTCAAGGTGTTAAAGGTGAATCTGGAAAGCCTGGAGCTAATGGTTTGTCAGGTGAAAGAGGACCTCCAGGTCCTCAAGGATTGCCTGGATTGGCTGGTACCGCTGGTGAGCCAGGTAGAGATGGTAACCCAGGTTCTGATGGATTGCCAGGTCGTGACGGATCCCCTGGTGGTAAAGGTGACAGAGGAGAAAACGGTTCTCCAGGTGCCCCTGGTGCTCCAGGACATCCAGGTCCACCAGGTCCAGTTGGTGAAAGAGGTTTTCCTGGTGAGAGAGGTGTTCAAGGACCAGCTGGACCAGCCGGAGCTCCAGGTCCAGCTGGATCTAGAGGAGCTCCTGGTCCTCAAGGTCCAAGAGGTGACAAAGGTGAAACCGGAGAGAGAGGTGCTGCCGGAATCAAGGGACATAGAGGTTTCCCAGGTAATCCAGGTGCTCCAGGTTCTCCTGGTCCAGCTGGACAACAAGGTGCCATTGGTTCTCCAGGACCAGCTGGTCCACGTGGTCCAGTTGGTCCATCCGGTCCACCAGGAAAGGATGGTACTTCCGGTCATCCAGGTCCAATCGGTCCACCAGGACCAAGAGGTAACAGAGGTGAGAGAGGTTCTGAAGGTTCTCCTGGTCATCCTGGTCAACCAGGTCCTCCAGGACCTCCTGGTGCTCCTGGTCCATGTTGTGGAGGTGTTGGTGCTGCAGCTATTGCTGGTATTGGTGGTGAAAAAGCTGGTGGTTTTGCTCCATACTATGGAGATGAGCCAATGGACTTTAAGATTAACACTGATGAAATTATGACATCTCTGAAATCTGTTAACGGACAAATTGAATCTTTGATTTCACCAGACGGTTCTAGAAAAAACCCAGCTAGAAATTGTAGAGACTTGAAGTTCTGTCATCCTGAGCTGAAATCTGGAGAATACTGGGTTGATCCTAACCAAGGTTGTAAGTTGGATGCTATTAAGGTTTTCTGTAATATGGAAACTGGTGAGACTTGTATTTCTGCTAACCCTCTGAATGTTCCAAGAAAGCATTGGTGGACTGACTCCTCCGCTGAAAAGAAGCACGTCTGGTTTGGTGAGTCTATGGATGGTGGTTTTCAGTTCTCATACGGTAACCCAGAGTTGCCAGAGGACGTTTTGGATGTTCACTTGGCTTTCTTGAGATTGTTGTCCTCTAGAGCTAGTCAAAACATTACCTACCATTGTAAAAACTCTATTGCTTATATGGACCAAGCTTCTGGTAATGTTAAGAAAGCTTTGAAGTTGATGGGTTCCAACGAGGGTGAGTTCAAGGCTGAGGGTAACTCTAAGTTTACTTACACTGTTTTGGAGGATGGTTGTACTAAGCATACTGGTGAGTGGTCTAAAACTGTTTTTGAATACAGAACTAGAAAGGCTGTTAGATTGCCAATTGTTGATATTGCTCCATACGATATCGGTGGTCCTGACCAAGAATTTGGAGTTGATGTTGGTCCAGTCTGTTTCTTG.

The present invention provides an expression vector comprising the nucleic acid molecule described above. The expression vector may contain regulatory sequences, such as transcription and translation initiation and termination codons, which are specific for the type of host to be introduced into the vector, e.g., bacterial, fungal, plant or animal, as appropriate and considering whether the vector is DNA-based or RNA-based. In a specific embodiment, the expression vector is pPIC9k, and the nucleotide sequence of the expression vector is shown as SEQ ID NO. 3.

SEQ ID NO.3:

AGATCTAACATCCAAAGACGAAAGGTTGAATGAAACCTTTTTGCCATCCGACATCCACAGGTCCATTCTCACACATAAGTGCCAAACGCAACAGGAGGGGATACACTAGCAGCAGACCGTTGCAAACGCAGGACCTCCACTCCTCTTCTCCTCAACACCCACTTTTGCCATCGAAAAACCAGCCCAGTTATTGGGCTTGATTGGAGCTCGCTCATTCCAATTCCTTCTATTAGGCTACTAACACCATGACTTTATTAGCCTGTCTATCCTGGCCCCCCTGGCGAGGTTCATGTTTGTTTATTTCCGAATGCAACAAGCTCCGCATTACACCCGAACATCACTCCAGATGAGGGCTTTCTGAGTGTGGGGTCAAATAGTTTCATGTTCCCCAAATGGCCCAAAACTGACAGTTTAAACGCTGTCTTGGAACCTAATATGACAAAAGCGTGATCTCATCCAAGATGAACTAAGTTTGGTTCGTTGAAATGCTAACGGCCAGTTGGTCAAAAAGAAACTTCCAAAAGTCGCCATACCGTTTGTCTTGTTTGGTATTGATTGACGAATGCTCAAAAATAATCTCATTAATGCTTAGCGCAGTCTCTCTATCGCTTCTGAACCCCGGTGCACCTGTGCCGAAACGCAAATGGGGAAACACCCGCTTTTTGGATGATTATGCATTGTCTCCACATTGTATGCTTCCAAGATTCTGGTGGGAATACTGCTGATAGCCTAACGTTCATGATCAAAATTTAACTGTTCTAACCCCTACTTGACAGCAATATATAAACAGAAGGAAGCTGCCCTGTCTTAAACCTTTTTTTTTATCATCATTATTAGCTTACTTTCATAATTGCGACTGGTTCCAATTGACAAGCTTTTGATTTTAACGACTTTTAACGACAACTTGAGAAGATCAAAAAACAACTAATTATTCGAAGGATCCAAACGATGAGATTTCCTTCAATTTTTACTGCAGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTCCAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTACTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTCTCGAGAAAAGAGAGGCTGAAGCTTACGTAGAATTCCCTAGGGCGGCCGCGAATTAATTCGCCTTAGACATGACTGTTCCTCAGTTCAAGTTGGGCACTTACGAGAAGACCGGTCTTGCTAGATTCTAATCAAGAGGATGTCAGAATGCCATTTGCCTGAGAGATGCAGGCTTCATTTTTGATACTTTTTTATTTGTAACCTATATAGTATAGGATTTTTTTTGTCATTTTGTTTCTTCTCGTACGAGCTTGCTCCTGATCAGCCTATCTCGCAGCTGATGAATATCTTGTGGTAGGGGTTTGGGAAAATCATTCGAGTTTGATGTTTTTCTTGGTATTTCCCACTCCTCTTCAGAGTACAGAAGATTAAGTGAGAAGTTCGTTTGTGCAAGCTTATCGATAAGCTTTAATGCGGTAGTTTATCACAGTTAAATTGCTAACGCAGTCAGGCACCGTGTATGAAATCTAACAATGCGCTCATCGTCATCCTCGGCACCGTCACCCTGGATGCTGTAGGCATAGGCTTGGTTATGCCGGTACTGCCGGGCCTCTTGCGGGATATCGTCCATTCCGACAGCATCGCCAGTCACTATGGCGTGCTGCTAGCGCTATATGCGTTGATGCAATTTCTATGCGCACCCGTTCTCGGAGCACTGTCCGACCGCTTTGGCCGCCGCCCAGTCCTGCTCGCTTCGCTACTTGGAGCCACTATCGACTACGCGATCATGGCGACCACACCCGTCCTGTGGATCTATCGAATCTAAATGTAAGTTAAAATCTCTAAATAATTAAATAAGTCCCAGTTTCTCCATACGAACCTTAACAGCATTGCGGTGAGCATCTAGACCTTCAACAGCAGCCAGATCCATCACTGCTTGGCCAATATGTTTCAGTCCCTCAGGAGTTACGTCTTGTGAAGTGATGAACTTCTGGAAGGTTGCAGTGTTAACTCCGCTGTATTGACGGGCATATCCGTACGTTGGCAAAGTGTGGTTGGTACCGGAGGAGTAATCTCCACAACTCTCTGGAGAGTAGGCACCAACAAACACAGATCCAGCGTGTTGTACTTGATCAACATAAGAAGAAGCATTCTCGATTTGCAGGATCAAGTGTTCAGGAGCGTACTGATTGGACATTTCCAAAGCCTGCTCGTAGGTTGCAACCGATAGGGTTGTAGAGTGTGCAATACACTTGCGTACAATTTCAACCCTTGGCAACTGCACAGCTTGGTTGTGAACAGCATCTTCAATTCTGGCAAGCTCCTTGTCTGTCATATCGACAGCCAACAGAATCACCTGGGAATCAATACCATGTTCAGCTTGAGACAGAAGGTCTGAGGCAACGAAATCTGGATCAGCGTATTTATCAGCAATAACTAGAACTTCAGAAGGCCCAGCAGGCATGTCAATACTACACAGGGCTGATGTGTCATTTTGAACCATCATCTTGGCAGCAGTAACGAACTGGTTTCCTGGACCAAATATTTTGTCACACTTAGGAACAGTTTCTGTTCCGTAAGCCATAGCAGCTACTGCCTGGGCGCCTCCTGCTAGCACGATACACTTAGCACCAACCTTGTGGGCAACGTAGATGACTTCTGGGGTAAGGGTACCATCCTTCTTAGGTGGAGATGCAAAAACAATTTCTTTGCAACCAGCAACTTTGGCAGGAACACCCAGCATCAGGGAAGTGGAAGGCAGAATTGCGGTTCCACCAGGAATATAGAGGCCAACTTTCTCAATAGGTCTTGCAAAACGAGAGCAGACTACACCAGGGCAAGTCTCAACTTGCAACGTCTCCGTTAGTTGAGCTTCATGGAATTTCCTGACGTTATCTATAGAGAGATCAATGGCTCTCTTAACGTTATCTGGCAATTGCATAAGTTCCTCTGGGAAAGGAGCTTCTAACACAGGTGTCTTCAAAGCGACTCCATCAAACTTGGCAGTTAGTTCTAAAAGGGCTTTGTCACCATTTTGACGAACATTGTCGACAATTGGTTTGACTAATTCCATAATCTGTTCCGTTTTCTGGATAGGACGACGAAGGGCATCTTCAATTTCTTGTGAGGAGGCCTTAGAAACGTCAATTTTGCACAATTCAATACGACCTTCAGAAGGGACTTCTTTAGGTTTGGATTCTTCTTTAGGTTGTTCCTTGGTGTATCCTGGCTTGGCATCTCCTTTCCTTCTAGTGACCTTTAGGGACTTCATATCCAGGTTTCTCTCCACCTCGTCCAACGTCACACCGTACTTGGCACATCTAACTAATGCAAAATAAAATAAGTCAGCACATTCCCAGGCTATATCTTCCTTGGATTTAGCTTCTGCAAGTTCATCAGCTTCCTCCCTAATTTTAGCGTTCAACAAAACTTCGTCGTCAAATAACCGTTTGGTATAAGAACCTTCTGGAGCATTGCTCTTACGATCCCACAAGGTGGCTTCCATGGCTCTAAGACCCTTTGATTGGCCAAAACAGGAAGTGCGTTCCAAGTGACAGAAACCAACACCTGTTTGTTCAACCACAAATTTCAAGCAGTCTCCATCACAATCCAATTCGATACCCAGCAACTTTTGAGTTGCTCCAGATGTAGCACCTTTATACCACAAACCGTGACGACGAGATTGGTAGACTCCAGTTTGTGTCCTTATAGCCTCCGGAATAGACTTTTTGGACGAGTACACCAGGCCCAACGAGTAATTAGAAGAGTCAGCCACCAAAGTAGTGAATAGACCATCGGGGCGGTCAGTAGTCAAAGACGCCAACAAAATTTCACTGACAGGGAACTTTTTGACATCTTCAGAAAGTTCGTATTCAGTAGTCAATTGCCGAGCATCAATAATGGGGATTATACCAGAAGCAACAGTGGAAGTCACATCTACCAACTTTGCGGTCTCAGAAAAAGCATAAACAGTTCTACTACCGCCATTAGTGAAACTTTTCAAATCGCCCAGTGGAGAAGAAAAAGGCACAGCGATACTAGCATTAGCGGGCAAGGATGCAACTTTATCAACCAGGGTCCTATAGATAACCCTAGCGCCTGGGATCATCCTTTGGACAACTCTTTCTGCCAAATCTAGGTCCAAAATCACTTCATTGATACCATTATTGTACAACTTGAGCAAGTTGTCGATCAGCTCCTCAAATTGGTCCTCTGTAACGGATGACTCAACTTGCACATTAACTTGAAGCTCAGTCGATTGAGTGAACTTGATCAGGTTGTGCAGCTGGTCAGCAGCATAGGGAAACACGGCTTTTCCTACCAAACTCAAGGAATTATCAAACTCTGCAACACTTGCGTATGCAGGTAGCAAGGGAAATGTCATACTTGAAGTCGGACAGTGAGTGTAGTCTTGAGAAATTCTGAAGCCGTATTTTTATTATCAGTGAGTCAGTCATCAGGAGATCCTCTACGCCGGACGCATCGTGGCCGACCTGCAGGGGGGGGGGGGGCGCTGAGGTCTGCCTCGTGAAGAAGGTGTTGCTGACTCATACCAGGCCTGAATCGCCCCATCATCCAGCCAGAAAGTGAGGGAGCCACGGTTGATGAGAGCTTTGTTGTAGGTGGACCAGTTGGTGATTTTGAACTTTTGCTTTGCCACGGAACGGTCTGCGTTGTCGGGAAGATGCGTGATCTGATCCTTCAACTCAGCAAAAGTTCGATTTATTCAACAAAGCCGCCGTCCCGTCAAGTCAGCGTAATGCTCTGCCAGTGTTACAACCAATTAACCAATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTCGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGGCTTTCCCCCCCCCCCCTGCAGGTCGGCATCACCGGCGCCACAGGTGCGGTTGCTGGCGCCTATATCGCCGACATCACCGATGGGGAAGATCGGGCTCGCCACTTCGGGCTCATGAGCGCTTGTTTCGGCGTGGGTATGGTGGCAGGCCCCGTGGCCGGGGGACTGTTGGGCGCCATCTCCTTGCATGCACCATTCCTTGCGGCGGCGGTGCTCAACGGCCTCAACCTACTACTGGGCTGCTTCCTAATGCAGGAGTCGCATAAGGGAGAGCGTCGAGTATCTATGATTGGAAGTATGGGAATGGTGATACCCGCATTCTTCAGTGTCTTGAGGTCTCCTATCAGATTATGCCCAACTAAAGCAACCGGAGGAGGAGATTTCATGGTAAATTTCTCTGACTTTTGGTCATCAGTAGACTCGAACTGTGAGACTATCTCGGTTATGACAGCAGAAATGTCCTTCTTGGAGACAGTAAATGAAGTCCCACCAATAAAGAAATCCTTGTTATCAGGAACAAACTTCTTGTTTCGAACTTTTTCGGTGCCTTGAACTATAAAATGTAGAGTGGATATGTCGGGTAGGAATGGAGCGGGCAAATGCTTACCTTCTGGACCTTCAAGAGGTATGTAGGGTTTGTAGATACTGATGCCAACTTCAGTGACAACGTTGCTATTTCGTTCAAACCATTCCGAATCCAGAGAAATCAAAGTTGTTTGTCTACTATTGATCCAAGCCAGTGCGGTCTTGAAACTGACAATAGTGTGCTCGTGTTTTGAGGTCATCTTTGTATGAATAAATCTAGTCTTTGATCTAAATAATCTTGACGAGCCAAGGCGATAAATACCCAAATCTAAAACTCTTTTAAAACGTTAAAAGGACAAGTATGTCTGCCTGTATTAAACCCCAAATCAGCTCGTAGTCTGATCCTCATCAACTTGAGGGGCACTATCTTGTTTTAGAGAAATTTGCGGAGATGCGATATCGAGAAAAAGGTACGCTGATTTTAAACGTGAAATTTATCTCAAGATCTCTGCCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGACCCAGTCACGTAGCGATAGCGGAGTGTATACTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTGCAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAACACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTTCAAGAATTAATTCTCATGTTTGACAGCTTATCATCGATAAGCTGACTCATGTTGGTATTGTGAAATAGACGCAGATCGGGAACACTGAAAAATAACAGTTATTATTCG.

The invention provides a sequence with hydroxylation function and collagen adhesion enhancement, and the amino acid sequence of the sequence is shown as SEQ ID NO. 4.

SEQ ID NO.4:

KYYEKIDGFLSDIECDVLINAAIKKGLIKSEVGGATENDPIKLDPKSRNSEQTWFMPGEHEVIDKIQKKTREFLNSKKHCIDKYNFEDVQVARYKPGQYYYHHYDGDDCDDACPKDQRLATLMVYLKAPEEGGGGETDFPTLKTKIKPKKGTSIFFWVADPVTRKLYKETLHAGLPVKSGEKIIANQWIRAVK.

The invention provides a sequence with hydroxylation function and enhanced collagen adhesion, which is used for codon optimization aiming at host cell expression, and signal peptide cleavage sites and EcoR I and Kpn I cleavage sites are respectively added at two ends in the design process so as to be beneficial to later gene operation. Through the optimization, the nucleotide sequence is shown as SEQ ID NO. 5.

SEQ ID NO.5:

ATGACCAACAAGTTCATCTCTTACAACAAGATGGAAACTCGTGAATACTTGCTGACCATTCTGTTCGTTATCGCTTGTTTCATGGTCTTGAACCTGGAAAGAAGAGAAGGTTTCGAGACTTCTGATAGACCAGGTGTTTGTGACGGTAAGTACTACGAGAAGATCGATGGATTTCTGTCTGACATCGAATGTGATGTGCTGATTAACGCTGCTATCAAGAAGGGTCTGATCAAGTCCGAAGTTGGTGGTGCTACTGAGAACGATCCAATCAAACTTGATCCAAAGTCTCGTAACTCCGAACAAACCTGGTTCATGCCAGGTGAACATGAGGTTATCGACAAGATCCAGAAGAAAACACGAGAGTTTCTGAACTCCAAGAAGCATTGCATCGACAAGTACAACTTCGAAGATGTTCAAGTTGCTAGGTACAAACCAGGTCAGTACTACTACCATCACTACGATGGTGATGATTGCGATGATGCTTGTCCAAAGGATCAGAGACTGGCTACTCTGATGGTTTACCTGAAGGCACCTGAAGAAGGTGGTGGAGGTGAAACCGATTTCCCTACTCTTAAGACCAAGATCAAGCCAAAGAAGGGTACTTCTATCTTCTTCTGGGTTGCTGATCCAGTCACCAGAAAGTTGTACAAAGAGACTTTGCATGCTGGTTTGCCAGTCAAGTCTGGTGAGAAGATTATCGCCAATCAGTGGATCAGAGCTGTTAAG.

The present invention provides an expression vector comprising the nucleic acid molecule described above. The expression vector may contain regulatory sequences, such as transcription and translation initiation and termination codons, which are specific for the type of host to be introduced into the vector, e.g., bacteria, fungi, plants or animals, and whether the vector is DNA-based or RNA-based as appropriate and considered. In a specific embodiment, the expression vector is pPICZA, the nucleotide sequence of which is shown in SEQ ID No. 6.

SEQ ID NO.6:

AGATCTAACATCCAAAGACGAAAGGTTGAATGAAACCTTTTTGCCATCCGACATCCACAGGTCCATTCTCACACATAAGTGCCAAACGCAACAGGAGGGGATACACTAGCAGCAGACCGTTGCAAACGCAGGACCTCCACTCCTCTTCTCCTCAACACCCACTTTTGCCATCGAAAAACCAGCCCAGTTATTGGGCTTGATTGGAGCTCGCTCATTCCAATTCCTTCTATTAGGCTACTAACACCATGACTTTATTAGCCTGTCTATCCTGGCCCCCCTGGCGAGGTTCATGTTTGTTTATTTCCGAATGCAACAAGCTCCGCATTACACCCGAACATCACTCCAGATGAGGGCTTTCTGAGTGTGGGGTCAAATAGTTTCATGTTCCCCAAATGGCCCAAAACTGACAGTTTAAACGCTGTCTTGGAACCTAATATGACAAAAGCGTGATCTCATCCAAGATGAACTAAGTTTGGTTCGTTGAAATGCTAACGGCCAGTTGGTCAAAAAGAAACTTCCAAAAGTCGGCATACCGTTTGTCTTGTTTGGTATTGATTGACGAATGCTCAAAAATAATCTCATTAATGCTTAGCGCAGTCTCTCTATCGCTTCTGAACCCCGGTGCACCTGTGCCGAAACGCAAATGGGGAAACACCCGCTTTTTGGATGATTATGCATTGTCTCCACATTGTATGCTTCCAAGATTCTGGTGGGAATACTGCTGATAGCCTAACGTTCATGATCAAAATTTAACTGTTCTAACCCCTACTTGACAGCAATATATAAACAGAAGGAAGCTGCCCTGTCTTAAACCTTTTTTTTTATCATCATTATTAGCTTACTTTCATAATTGCGACTGGTTCCAATTGACAAGCTTTTGATTTTAACGACTTTTAACGACAACTTGAGAAGATCAAAAAACAACTAATTATTCGAAACGAGGAATTCACGTGGCCCAGCCGGCCGTCTCGGATCGGTACCTCGAGCCGCGGCGGCCGCCAGCTTGGGCCCGAACAAAAACTCATCTCAGAAGAGGATCTGAATAGCGCCGTCGACCATCATCATCATCATCATTGAGTTTTAGCCTTAGACATGACTGTTCCTCAGTTCAAGTTGGGCACTTACGAGAAGACCGGTCTTGCTAGATTCTAATCAAGAGGATGTCAGAATGCCATTTGCCTGAGAGATGCAGGCTTCATTTTTGATACTTTTTTATTTGTAACCTATATAGTATAGGATTTTTTTTGTCATTTTGTTTCTTCTCGTACGAGCTTGCTCCTGATCAGCCTATCTCGCAGCTGATGAATATCTTGTGGTAGGGGTTTGGGAAAATCATTCGAGTTTGATGTTTTTCTTGGTATTTCCCACTCCTCTTCAGAGTACAGAAGATTAAGTGAGACCTTCGTTTGTGCGGATCCCCCACACACCATAGCTTCAAAATGTTTCTACTCCTTTTTTACTCTTCCAGATTTTCTCGGACTCCGCGCATCGCCGTACCACTTCAAAACACCCAAGCACAGCATACTAAATTTTCCCTCTTTCTTCCTCTAGGGTGTCGTTAATTACCCGTACTAAAGGTTTGGAAAAGAAAAAAGAGACCGCCTCGTTTCTTTTTCTTCGTCGAAAAAGGCAATAAAAATTTTTATCACGTTTCTTTTTCTTGAAATTTTTTTTTTTAGTTTTTTTCTCTTTCAGTGACCTCCATTGATATTTAAGTTAATAAACGGTCTTCAATTTCTCAAGTTTCAGTTTCATTTTTCTTGTTCTATTACAACTTTTTTTACTTCTTGTTCATTAGAAAGAAAGCATAGCAATCTAATCTAAGGGGCGGTGTTGACAATTAATCATCGGCATAGTATATCGGCATAGTATAATACGACAAGGTGAGGAACTAAACCATGGCCAAGTTGACCAGTGCCGTTCCGGTGCTCACCGCGCGCGACGTCGCCGGAGCGGTCGAGTTCTGGACCGACCGGCTCGGGTTCTCCCGGGACTTCGTGGAGGACGACTTCGCCGGTGTGGTCCGGGACGACGTGACCCTGTTCATCAGCGCGGTCCAGGACCAGGTGGTGCCGGACAACACCCTGGCCTGGGTGTGGGTGCGCGGCCTGGACGAGCTGTACGCCGAGTGGTCGGAGGTCGTGTCCACGAACTTCCGGGACGCCTCCGGGCCGGCCATGACCGAGATCGGCGAGCAGCCGTGGGGGCGGGAGTTCGCCCTGCGCGACCCGGCCGGCAACTGCGTGCACTTCGTGGCCGAGGAGCAGGACTGACACGTCCGACGGCGGCCCACGGGTCCCAGGCCTCGGAGATCCGTCCCCCTTTTCCTTTGTCGATATCATGTAATTAGTTATGTCACGCTTACATTCACGCCCTCCCCCCACATCCGCTCTAACCGAAAAGGAAGGAGTTAGACAACCTGAAGTCTAGGTCCCTATTTATTTTTTTATAGTTATGTTAGTATTAAGAACGTTATTTATATTTCAAATTTTTCTTTTTTTTCTGTACAGACGCGTGTACGCATGTAACATTATACTGAAAACCTTGCTTGAGAAGGTTTTGGGACGCTCGAAGGCTTTAATTTGCAAGCTGGAGACCAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATC.

The present invention provides a host cell comprising the above-described nucleic acid molecule.

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

In a specific embodiment, the host cell is selected from any one of pichia pastoris, saccharomyces cerevisiae, escherichia coli, bacillus subtilis.

In a specific embodiment, the host cell is pichia pastoris GS115.

The invention provides a method for preparing any one of the recombinant collagen, which comprises the following steps:

The expression of the host cells is performed by using the above-mentioned host cells, followed by isolation and purification, and the expression of the host cells means that the host cells are cultured, and the culture medium and the culture conditions are well known to those skilled in the art.

In a specific embodiment, the host cell is Pichia pastoris, after the Pichia pastoris genetic engineering bacteria are obtained, the specific culture conditions are that the Pichia pastoris genetic engineering bacteria are inoculated into YPD culture medium, cultured for 22-24 hours under the conditions of 30 ℃ and 220rpm until OD600 is 18-20 as seed liquid of an upper tank, the seed liquid is inoculated into an NBS 415 fermentation tank with an initial volume of 5L according to 10% of the inoculation amount of the seed liquid after the seed liquid is expanded, the culture temperature is 28-30 ℃ and the pH value is 5.0-6.0, the dissolved oxygen is controlled to be 20-30%, and after glycerol is exhausted, the culture of glycerol feed supplement is started until the wet weight of the thallus reaches more than 180 g/L.

The expression pattern is not limited in any way, and it can be confirmed as needed, for example, expression as induced expression, and for induced expression, the inducer methanol.

In a specific embodiment, methanol is fed for induction culture, the temperature of the induction stage is 28 ℃, the pH is 5.0, and the induction is carried out for 48 hours.

The method for separation and purification is not limited in any way, and may be determined according to the implementation, and for example, salting out, ultrafiltration, affinity chromatography, and gel filtration chromatography may be used.

The invention also provides the recombinant hydroxylated collagen, or the recombinant collagen encoded by the nucleic acid molecule, or the recombinant hydroxylated collagen expressed by the expression vector, or the recombinant hydroxylated collagen produced by the host cell, which has the application of immune response, inflammatory response, coagulation, tumor metastasis and wound healing.

EXAMPLE 1 expression of recombinant hydroxylated human type III collagen

The recombinant collagen gene of the invention, namely the target gene, is chemically synthesized, and the nucleotide sequence is shown as SEQ ID NO. 2. EcoR I and Not I recognition sites and signal peptide recognition sites are respectively added at the 5 'end and the 3' end during synthesis, the cloning is carried out on the cloning vector pPIC9K after linearization by restriction enzyme Sac I, so as to obtain pPIC9K-RCOL (III) cloning plasmid, pichia pastoris GS115 is taken as an expression host bacterium, the obtained pPIC9K-RCOL (III) cloning plasmid is linearized by electric conversion and then is converted into GS115 to be GS115-pPIC9K-RCOL (III), high-copy positive cloning is selected by G418 gradient method, and pichia pastoris genetic engineering bacteria is obtained after 30 ℃ culture for 72 hours. The linearity results of the pPIC9K-RCOL (III) cloning plasmid are shown in FIG. 1A.

Chemical synthesis the hydroxylation gene of the invention has a nucleotide sequence shown in SEQ ID NO. 5. EcoR I and Kpn I recognition sites and signal peptide recognition sites are respectively added at the 5 'end and the 3' end during synthesis, and the vector pPICZA-PH4 is obtained by cloning the vector into an expression vector pPICZA after linearization of restriction enzyme Sac I. The pichia pastoris GS115-pPIC9K-RCOL (III) is taken as an expression host bacterium, the obtained pPICZA-PH4 cloning plasmid is linearized through electric transformation, and then is transformed into a recombinant strain GS115-pPIC9K-RCOL (III) to be GS115-pPIC9K-RCOL (III) -pPICZA-PH4, and the recombinant strain GS115-pPIC9K-RCOL (III) is screened by bleomycin. The results of pPICZA-PH4 pellet linearization are shown in FIG. 1B.

Screening positive transformants, namely selecting a few transformants in high-copy positive clones by a G418 gradient method, selecting a few transformants by a marker pen, coating a small amount of thalli of the selected transformants on the bottom of a PCR tube, adding 50 mu L of enzyme-free water, blowing and mixing uniformly, heating at a medium high temperature in a microwave oven for 10min, immediately putting into a refrigerator at-80 ℃ for 10min, repeating for 5 times, centrifuging at 12000rpm for 1min, taking 3 mu L of supernatant as a template for PCR reaction, and screening GS115-pPIC9K-RCOL (III) -pPICZA-PH4 by zeo ⁺. The agarose gel electrophoresis patterns of GS115-pPIC9K-RCOL (III) and GS115-pPIC9K-RCOL (III) -pPICZA-PH4 are shown in FIGS. 2 and 3.

Inoculating the obtained Pichia pastoris genetic engineering bacteria into YPD culture medium, culturing until OD600 is 19.88, inoculating into NBS 415 fermentation tank with initial volume of 5L according to 10% of the volume of the inoculated strain, controlling the culture temperature to 30 ℃ and pH to 5.5, controlling dissolved oxygen to 20%, starting glycerol feeding culture after glycerol is exhausted, starting adding methanol until the thallus wet weight reaches more than 190g/L, performing induction culture at a methanol flow rate of 80mL/h, performing induction stage temperature of 28 ℃, pH to 5.0, discharging the tank after 48h induction, and centrifuging to collect supernatant.

EXAMPLE 2 purification of recombinant hydroxylated human type III collagen

1. When the supernatant collected by centrifugation was ultrafiltered to 50% of the initial volume, 5 volumes of pure water was added, and the mixture was concentrated to 5% of the initial volume by ultrafiltration.

2. The concentrated supernatant was added with saturated ammonium sulfate in an amount of 60% of the volume of the concentrated supernatant, stirred at room temperature for 30min, centrifuged at 9000rpm for 10min, and the precipitate was collected, dissolved in 500ml of 0.05m PBS, ph 7.0, and filtered through a 0.22 μm filter membrane.

3. According to isoelectric point of the protein, preparing equilibrium buffer solution, namely 20mmol/L sodium phosphate buffer solution with pH of 6.0, namely solution A, and preparing eluent with pH of 6.0 by mixing sodium phosphate with molar mass of 20mmol/L and NaCl with molar mass of 1.0mol/L, namely solution B. Diluting the PBS protein solution obtained in the last step by using the solution A in a ratio of 10:1 to prepare a loading solution, filtering, loading the loading solution on a 25mL CM-Sepharose cation exchange chromatography column, and balancing the column by using a balancing buffer solution before loading. After the sample loading is finished, 2 column volumes are washed by the solution A, and then gradient elution is carried out by the solution A with the concentration of 70% and the solution B with the concentration of 30% and the flow rate of 2mL/min. The eluted fractions were collected and examined by SDS-PAGE.

4. According to the distribution range of the molecular weight of the protein obtained after ion exchange chromatography, a Sephadex 200 gel column is selected to further purify the target protein. The AKTA operation process comprises washing with balance buffer solution (PBS) 0.01mol/L and NaCl 0.05mol/L until baseline is stable, loading recombinant GS115-pPIC9K-RCOL (III) -pPICZA-PH4 protein component eluted by ion exchange column obtained in the previous step onto gel filtration chromatographic column filled with Superdex 200, eluting with eluent, setting flow rate to 10mL/min, and ultraviolet detection wavelength to 215nm. Finally, the effluent target protein fraction was collected after SDS-PAGE electrophoresis.

5. The method comprises the steps of ultrafiltration desalination and G25 desalination column desalination, namely adopting 25mL of G25 packing, wherein the operation process is similar to that of gel filtration chromatography, each time of sample loading is 6.5mL, about 8mL of sample is collected, and the desalination can be completed after sample loading is carried out for 10 min.

6. Concentrating to 30% of the initial volume by ultrafiltration, pre-freezing in a refrigerator at-20 ℃ for 4 hours, then transferring into a vacuum freeze dryer for freeze-drying, collecting freeze-dried protein after 48 hours, and storing the freeze-dried protein sample in the refrigerator at-80 ℃ for later use.

EXAMPLE 3 use of recombinant hydroxylated human type III collagen in the preparation of cell adhesion promoter

1. Cell adhesion experiments

Two 48-well plates were coated with 300 μl of bovine serum albumin BSA, native human type III collagen, recombinant human type III collagen, and recombinant hydroxylated human type III collagen solution at a concentration of 0.2mg/mL, respectively, overnight at 4 ℃, blocked with 1% heat denatured BSA by mass for 1h at room temperature, and then washed twice with PBS. L929 cells in the logarithmic growth phase were diluted and counted, and finally cell suspensions with cell densities of 2.0X10 ⁵ cells/mL were inoculated on 48-well plates coated with different proteins, and the two well plates were cultured in CO ₂ biochemical incubator for 4h and 24h, respectively.

After 4h incubation, the culture supernatant was discarded, the L929 cells were washed three times with PBS, a mass fraction of 1% SDS solution was added to the well plate, and the OD value thereof was measured at 590nm, and the control group was the total number of cells washed without PBS. Cell adhesion was calculated using the formula:

Cell adhesion rate=[(remaining cell OD590)/(Control cell OD590)]

after 24h incubation, L929 cells were washed three times with PBS, the remaining cells were fixed with 4% paraformaldehyde for 20 minutes and stained with 0.1% crystal violet by mass for 20 minutes, after which the cell morphology was observed under an optical microscope and analyzed.

The relative adhesion rate of cells was calculated from the absorbance values, BSA was set to 100%, the cell adhesion chart was shown in FIG. 4, and the cell adhesion rate statistics were shown in Table 1

2. Cell proliferation-promoting experiment

L929 cells in the logarithmic growth phase were seeded on 96-well tissue culture plates and cultured for 1d at 37℃under a 5% volume fraction CO ₂ biochemical incubator. The old medium was discarded, BSA, native human type III collagen, recombinant human type III collagen and recombinant hydroxylated human type III collagen were sequentially dissolved in DMEM medium at a concentration of 0.2mg/ml, filtered and sterilized into the above 96-well plates. After 24h incubation, all incubated L929 cells were stained with AO/EB and their growth after incubation in collagen medium was observed under a fluorescence microscope, and the AO/EB staining pattern is shown in FIG. 5.

	BSA	Natural type III collagen	Recombinant type III collagen	Recombinant hydroxylated type III collagen
					Adhesion rate	100%	109%	126%	136%

The result shows that the III type recombinant hydroxylated collagen can obviously promote the adhesion of L929 cells, has higher adhesion promoting effect than natural collagen, is obviously higher than BSA, has good cell compatibility and better promotion effect, and is consistent with the observation result of a light microscope.

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention.

Claims

1. A recombinant human type III collagen with cell adhesion, characterized in that the amino acid sequence is shown in SEQ ID NO.1.

2. A gene encoding the recombinant human type III collagen as claimed in claim 1, characterized in that the nucleotide sequence is as shown in SEQ ID NO.2.

3. A sequence having a hydroxylation function and enhancing the adhesion of the recombinant human type III collagen as claimed in claim 1, characterized in that the amino acid sequence is as shown in SEQ ID NO.4 and the nucleotide sequence is as shown in SEQ ID NO.5.

4. The use of the recombinant human type III collagen according to claim 1, characterized in that the recombinant human type III collagen is used to prepare a cell adhesion promoter.

5. An expression vector, characterized in that it comprises the nucleotide sequence according to claim 2.

6. An expression vector, characterized in that it comprises the nucleotide sequence according to claim 3.

7. A host cell, characterized in that it comprises the expression vector according to any one of claim 5 or claim 6.

8. The host cell according to claim 7, characterized in that the host cell is any one of Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli and Bacillus subtilis.

9. A method for preparing recombinant human type III collagen using the host cell of claim 7, characterized in that the host cell is cultured in a culture medium, the expression of the recombinant human type III collagen is induced, and then the recombinant human type III collagen is purified to obtain the recombinant human type III collagen.

10. The method according to claim 9, characterized in that the purification method is selected from any one of salting-out, ultrafiltration, affinity chromatography and gel filtration chromatography.