AU2016101562A4 - Genetic recombinant human collagen, gene encoding the same, and preparation method thereof - Google Patents

Genetic recombinant human collagen, gene encoding the same, and preparation method thereof Download PDF

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AU2016101562A4
AU2016101562A4 AU2016101562A AU2016101562A AU2016101562A4 AU 2016101562 A4 AU2016101562 A4 AU 2016101562A4 AU 2016101562 A AU2016101562 A AU 2016101562A AU 2016101562 A AU2016101562 A AU 2016101562A AU 2016101562 A4 AU2016101562 A4 AU 2016101562A4
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human collagen
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En GAO
Zengmiao Hou
Min Li
Xiaoying Li
Xiaolin Yang
Jinli Zhao
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Revolution Era Holdings Pty Ltd
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    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

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Abstract

The present invention provides a recombinant human collagen, Also provided a preparation method for said recombinant human collagen, composing the following steps: ( preparation of plasmids: synthesizing the sequence of SEQ I D No: 3 in the sequence list by artifiial total gene synthesis, subjecting the pPIC9K vector and the human collagen synthesized by artificial total gene synthesis to double digestion with enzymes Xho I and EcoR I, then extracting the plasmids; (2) transformation: mixing the prepared plasmids in step () with Pichia pastoris for transformation, obtaining transformed bacterial colonies (3) screening the multi-copy inserted recombinant; (4) fermenting the rnuhi-copy hiserted recombinant to obtain fermentation broth; (5) purification of the fermentation broth to give the product. The present invention also provides a gene sequence encoding the recombinant human collagen, The human collagen according to the present invention has excellent biocompatibiliy, and the purity of the collagen is 95% or more. Figure 1 Drawing Z Cx nRQ9K-NOL .. Figure I

Description

GENETIC RECOMBINANT HUMAN COLLAGEN, GENE ENCODING THE SAME, AND PREPARATION METHOD THEREOF
Technical Field
The present invention belongs to the field of bioengineering, and particularly relates to a genetic recombinant human collagen and coding gene thereof, the present invention also relates to a preparation method of the recombinant human collagen.
Background of the Invention
Collagen, which is about 25% -33% by the total amount of proteins, is a most abundant protein in the body, and widely present in bones, tendons, skins and cartilages and other connective tissues. Collagen is a main ingredient of the extracellular matrix (ECM), and plays an important role in maintaining the normal physiological functions of cells, tissues and organs, and repairing damages. From the point of molecular structure, collagen is composed of parallel linear chains, and each of the linear chains has a strong dextrorotatory triple helical structure formed by three distorted levorotatory a-peptide chains bonded closely through interactions among the chains. Each α-peptide chain is made up of 300 or more Gly-X-Y triplets, and both ends of each α-peptide chain are bonded by other small segments having different structures. The amino acid residues which form collagen are α-amino acids, wherein X and Y are any amino acid residues other than Gly.
As a natural biological resource, collagen has much better biocompatibility and biodegradability than synthetic polymer materials, and can be widely used in medicine, health products and cosmetics industries. In prior arts, a traditional and most important process of preparing collagen is extracting collagen by treating tissues (such as pigskin, cowhide, donkey hide, fish, etc.) of animals with acids or bases. In addition, collagen also can be extracted with enzymes. Although high recovery rates have been reached by these methods, the collagens prepared by these methods are a mixture of collagen peptides with different lengths, the water solubility of which are different, so that it is difficult to obtain pure isolated peptides. Further, the collagens are heterologous collagens, and exhibit rejection reactions clinically, which greatly influences the application of the collagens as biomedical materials, drug carriers or the like.
With the development of modern biotechnology, recombinant human collagen may be obtained from the expression systems of animals, plants and microorganism by using transgenic technology and gene recombination technology, which not only solves the defects such as the threat of viruses, etc., existed in the conventional extraction methods, but also improves the hydrophilicity, immune exclusion and the like of collagen. Some domestic and foreign research institutions and biotech companies have invested in the development of recombinant collagen. High expression is achieved in E. coli by repeating type ΠΙ collagen peptides and fusing the type III collagen peptides with type II collagen peptides (YANG Xia et al., 2012). Human-like collagens with high expression are produced by high-density fermentation cultivation of E. coli, and are used as raw materials for development of tissue engineering materials and cosmetics (FAN Daidi, et al., Northwestern University, China). However, a part of the amino acid sequences designed is not human-derived collagen sequences, while pyrogen expressed by bacterium makes it difficult to apply the expressed products clinically. Also, the target protein is often expressed in the form of inclusion body, resulting in difficulty of purifying products. Further, the defects of prokaryotic expression system, such as the imperfect post-translational modification and processing system, and the low biological activity of the product, deteriorate the quality of the product. Therefore, in the technical field of preparing human collagen, there is an urgent need to develop a human collagen with high purity, high security, strong hydrophilicity and good biocompatibility, and a method by which the expression may not be performed in bacterium.
Disclosure of the Invention
The technical problem to be solved by the present invention is to provide a genetic recombinant human collagen and genes encoding the same.
The present invention provides a technical solution with respect to the deficiencies in the prior art: existing collagens, which are structurally heterologous, are usually a mixture of peptides with different lengths; since the water solubility of the peptides are different, it is difficult to obtain pure isolated peptides. For the aforementioned reasons, the existing collagens likely exhibit rejection reactions clinically, which greatly influences the application of the collagens in clinical.
In particular, one technical problem to be solved by the present invention is to provide a genetic recombinant human collagen with excellent water solubility, high expression quantity and high purity.
Another technical problem to be solved by the present invention is to provide a simple and convenient preparation method for the recombinant human collagen.
Another technical problem to be solved by the present invention is to provide genes encoding the recombinant human collagen.
More specifically, the above technical problems are solved by the following technical solutions . A recombinant human collagen, wherein the collagen comprises one or more of the following family members: 1) protein having the amino acid sequence of SEQ ID No: 1 in the sequence list; 2) SEQ ID No: 1 derived protein, having a sequence homology of 90% or more to the amino acid sequence of SEQ ID No: 1 and having the same activity as that of SEQ ID No: i; 3) SEQ ID No: 1 derived protein, having the same activity as that of SEQ ID No: 1, obtained by addition or deletion of 15 or less amino acid residues at N-terminal of the amino acid sequence of SEQ ID No: 1; 4) SEQ ID No: 1 derived protein, having the same activity as that of SEQ ID No: 1, obtained by addition or deletion of 15 or less amino acid residues at C-terminal of the amino acid sequence of SEQ ID No: 1; 5) SEQ ID No: 1 derived protein, having the same activity as that of SEQ ID No: 1, obtained by substitution, deletion or addition of one or more amino acid residues in the amino acid residue sequence of SEQ ID No: 1.
Wherein, the protein is SEQ ID No: 1 in the sequence list.
Wherein, the isoelectric point of the protein is 9.5.
Wherein, the host cell for expressing the protein is Pichia. Pastoris. A preparation method for the recombinant human collagen, wherein the method comprises the following steps: (1) preparation of plasmids: synthesizing the sequence of SEQ ID No: 3 in the sequence list by artificial total gene synthesis, subjecting the pPIC9K vector and the human collagen of SEQ ID No:3 synthesized by artificial total gene synthesis to double digestion with enzymes Xho I and EcoR I, recovering the enzyme-digested product, ligating the product by using DNA ligase, transforming the ligation product into Colon bacillus, and extracting plasmids; (2) transformation: mixing the prepared plasmids in step (1) with Pichia pastoris for transformation, and obtaining transformed colonies; (3) screening the multi-copy inserted recombinant: cultivating the transformed colonies obtained in step (2), and screening transformants; (4) fermentation: inoculating and fermenting the screened transformants in step (3) to obtain fermentation broth; (5) purification: successively subjecting the fermentation broth in step (4) to solid-liquid separation, filtration, concentration, and ion-exchange column chromatography to obtain the product.
Wherein, the purification in step (5) comprises: subjecting the fermentation broth obtained in step (4) to solid-liquid separation to collect the supemate; subjecting the supernate to microfiltration by a hollow fiber microfiltration system with a pore diameter of 0.22 pm, collecting the filtrate; subjecting the filtrate to desalination and concentration by ultrafiltration with a hollow fiber ultrafiltration system with a molecular weight cutoff of 10 KD, collecting the concentrated solution; then subjecting the concentrated solution to ion-exchange column chromatography with CM Sepharose FF to obtain the product. Wherein, the method comprises the following steps: (1) preparation of plasmids synthesizing the sequence of SEQ ID No: 3 in the sequence list by artificial total gene synthesis, subjecting the pPIC9K vector and the human collagen of SEQ ID No:3 synthesized by artificial total gene synthesis to double digestion with enzymes Xho I and EcoR I simultaneously, recovering the enzyme-digested product, ligating the product by using DNA ligase, transform the ligation product into Colon bacillus, and extracting the plasmids referred to as pPIC9K-NCOL; (2) transformation of Pichia pastoris mixing the pPIC9K-NCOL plasmid linearized by Sal I endonuclease with Pichia pastoris competent cells, transferring the mixture into a ice-precooled electroporation cup, subjecting the mixture to electrical shock for 4-10 milliseconds, adding ice-precooled sorbitol solution therein and mixing, spreading the resulting mixture on a MD medium plate, then culturing inverted MD medium plate for 2-3 days, such that bacterial colonies grow on the MD medium plate; (3) screening multi-copy inserted recombinant correspondingly inoculating the bacterial colonies on the MD medium plate with sterile toothpicks to YPD plates with G418 concentrations of 0.5 g/L, 1 g/L, 2 g/L, 3 g/L, 4 g/L, and 5 g/L separately, cultivating the YPD plates, then screening to obtain the transformants; (4) fermentation of genetic recombinant human collagen inoculating the screened transformants to BMGY medium, and subjecting the inoculated BMGY medium to shaking cultivation for 24 hours, inoculating the cultivated BMGY medium as the first order seed to a fermentation tank loaded with FBS medium to cultivate at a pH of 5.0 for 16-20 hours, then inoculating the cultivated FBS medium as the second order seed to a fermentation tank loaded with FBS medium to perform an induction fermentation for 36-42 hours, with a growth temperature of 30 °C, an inducing temperature lower than the growth temperature, a pH of 5.0, and a dissolve oxygen in a range of 20%-30%; (5) purification of genetic recombinant human collagen subjecting the fermentation broth in step (4) to solid-liquid separation to give supemate; subjecting the supernate to microfiltration by a hollow fiber microfiltration system with a pore diameter of 0.1 pm or 0.22 pm, collecting the filtrate; subjecting the filtrate to desalination and concentration by ultrafiltration with a hollow fiber ultrafiltration system with a molecular weight cutoff of 10 KD, collecting the concentrated solution; then subjecting the concentrated solution to ion-exchange column chromatography with CM Sepharose FF to obtain the genetic recombinant human collagen with a purity of 95% or more. A gene encoding a recombinant human collagen, the gene is one of the following nucleotide sequences: 1) SEQ ID No: 2 in the sequence list; 2) polynucleotides encoding the protein sequence of SEQ ID No:l in the sequence list; 3) DNA sequence, which has a homology of 90% or more to the DNA sequence defined by SEQ ID No: 2 in the sequence list and encodes the protein with the same function as that of the protein encoded by SEQ ID No: 2; 4) DNA sequence encoding the protein derived from SEQ ID No: 1, the protein derived from SEQ ID No: 1 is a polypeptide having the same activity as that of SEQ ID No: 1, obtained by addition or deletion of 15 or less amino acid residues at N-terminal of the amino acid sequence of SEQ ID No: 1; 5) DNA sequence encoding the protein derived from SEQ ID No: 1, the protein derived from SEQ ID No: 1 is a polypeptide having the same activity as that of SEQ ID No: 1, obtained by addition or deletion of 15 or less amino acid residues at C-terminal of the amino acid sequence of SEQ ID No: 1; 6) DNA sequence coding the protein derived from SEQ ID No: 1, the protein derived from SEQ ED No: 1 is a polypeptide having the same activity as that of SEQ ID No: 1, obtained by substitution, deletion or addition of one or more amino acid residues in the amino acid sequence of SEQ ID No: 1.
Wherein, the gene is SEQ ID No: 2 in the sequence list.
An expression vector containing the gene. A cell line containing the gene. A biological material for tissue engineering and skincare, the biological material contains the human collagen or the recombinant human collagen encoded by the gene.
The present invention further provides the recombinant human collagen, the recombinant human collagen prepared by the method, and the recombinant human collagen encoded bythe gene, wherein the purity of the collagen is 95% or more.
The advantages of the present invention lie in that:
The collagen obtained according to the present invention has the characters of high expression quantity, excellent water-solubility and high purity, which lay the foundation for the application of genetic recombinant human- source collagen.
According to the present invention, hydrophilic Gly-X-Y in human collagen is preferably permuted and combined. The Gly-X-Y has the same sequence as that of human collagen; biocompatibility of the Gly-X-Y is good, and the purity of the Gly-X-Y is more than 95%.
Brief Description of the Drawings
Figure 1 shows a plasmid profile of the recombinant expression vector pPIC9K-NCOL according to Example 1.
Figure 2 shows a SDS-PAGE image of fermentation supernatant of genetic recombinant human collagen and the purified sample according to Example 1.
Figure 3 shows a cell proliferation curve according to Example 1.
Figure 4 shows an image of the influences of different materials on cell morphology according to Example 1.
Specific Mode for Carrying out the Present Invention
In order to explain the technical solutions adopted to solve the technical problems of the present invention, the present invention will be further explained and described with reference to the following examples and figures, but the technical solutions, the modes for carrying out the present invention and the protection scope are not limited thereto.
The present invention provides a recombinant human collagen, the collagen is composed of human collagen type I Gly-X-Y repetitive sequence as a minimum repeating unit, wherein X, Y are amino acids respectively. The human collagen has a structure that Gly-X-Y repetitive sequence serves as the minimum repeat unit. Based on that, hydrophilic Gly-X-Y in human collagen is preferably permuted and combined according to the present invention.
In one embodiment, the Gly-X-Y is a hydrophilic amino acid chain; the collagen comprises one or more of the following family members: 1) protein having the amino acid sequence of SEQ ID No: 1 in the sequence list; 2) SEQ ID No: 1 derived protein, having a sequence homology of 90% or more to the amino acid sequence of SEQ ID No: 1 and having the same activity as that of SEQ ID No: i; 3) SEQ ID No: 1 derived protein, having the same activity as that of SEQ ID No: 1, obtained by addition or deletion of 15 or less amino acid residues at N-terminal of the amino acid residue sequence of SEQ ID No: 1; 4) SEQ ID No: 1 derived protein, having the same activity as that of SEQ ID No: 1, obtained by addition or deletion of 15 or less amino acid residues at C-terminal of the amino acid residue sequence of SEQ ID No: 1; 5) SEQ ID No: 1 derived protein, having the same activity as that of SEQ ID No: 1, obtained by substitution, deletion or addition of one or more amino acid residues in the amino acid residue sequence of SEQ ID No: 1.
The technical solution known by the person skilled in the art that the basic structure of the protein according to the present invention is appropriately added or reduced by a certain amount of amino acid sequences so that the resulting protein may have the same activities as that of above protein, also comes into the protection scope of the present invention. The certain amount according to the present invention is 15, since less than 15 amino acid sequences are hard to form a protein having a specific activity. That is, under the technical concept of the present invention, any collagen obtained by using hydrophilic Gly-XY repeating sequence as the minimum repeating unit may come into the protection scope of the present invention.
In one embodiment, the protein is SEQ ID No: 1 in the sequence list; the isoelectric point of the protein is 9.5, which facilitates purification later; the host cell for expressing the protein is Pichia. Pastoris.
In one embodiment, the present invention also provides a preparation method for said recombinant human collagen, and the method comprises the following steps: (1) preparation of plasmids: synthesizing the sequence of SEQ ID No: 3 in the sequence list by artificial total gene synthesis, subjecting the pPIC9K vector and the human collagen of SEQ ID No:3 synthesized by artificial total gene synthesis to double digestion with enzymes Xho I and EcoR I, recovering the enzyme-digested product, ligating the product by using DNA ligase, transforming the ligation product into Colon bacillus, and extracting the plasmids;
In step (1), synthesizing the sequence of SEQ ID No: 3 in the sequence list by artificial total gene synthesis comprises designing and synthesizing the amide acid sequences of the genetic recombinant human collagen by artificial total gene synthesis in accordance with the codon bias of Pichia. Pastoris, based on the designed amide acid sequences of the genetic recombinant human collagen; meanwhile, cleavage site CTCGAG of the Xho I endonuclease and signal peptide cleavage site sequence AAAAGA of Pichia. Pastoris are added at 5' end, and cleavage site GAATTC of the EcoR I endonucleas is added at 3' end; (2) transformation: mixing the prepared plasmids in step (1) with Pichia pastoris for transformation, and obtaining transformed bacterial colonies; (3) screening the multi-copy inserted recombinant: cultivating the transformed bacterial colonies obtained in step (2), and screening transformants; (4) fermentation: inoculating and fermenting the screened transformants in step (3) to obtain fermentation broth; and (5) purification: successively subjecting the fermentation broth in step (4) to solid-liquid separation, filtration, concentration, and ion-exchange column chromatography to obtain the product.
In one preferable embodiment, the purification in step (5) comprises the steps of: subjecting the fermentation broth in step (4) to solid-liquid separation to give supemate; subjecting the supemate to microfiltration by a hollow fiber microfiltration system with a pore diameter of 0.22 pm, collecting the filtrate; subjecting the filtrate to desalination and concentration by ultrafiltration with a hollow fiber ultrafiltration system with a molecular weight cutoff of 10 KD, collecting the concentrated solution; then subjecting the concentrated solution to ion-exchange column chromatography with CM Sepharose FF to obtain the product.
In one preferable embodiment, the method comprises the following steps: (1) preparation of plasmids synthesizing the sequence of SEQ ID No: 3 in the sequence list by artificial total gene synthesis, subjecting the pPIC9K vector and the human collagen of SEQ ID No:3 synthesized by artificial total gene synthesis to double digestion with enzymes Xho I and EcoR I, recovering the enzyme-digested product, ligating the product by using DNA ligase, transforming the ligation product into Colon bacillus, and extracting the plasmids referred to as pPIC9K-NCOL; (2) transformation by Pichia pastoris mixing the plasmids linearized by Sal I endonuclease with Pichia pastoris competent cells, transferring the mixture into a ice-precooled electroporation cup, subjecting the mixture to electrical shock for 4-10 milliseconds, adding ice-precooled sorbitol solution thereto, spreading the resulting mixture on a MD medium plate, then culturing inverted MD medium plate for 2-3 days, such that bacterial colonies grow on the MD medium plate; (3) screening the multi-copy inserted recombinant correspondingly inoculating the bacterial colonies on the MD medium plate with sterile toothpicks to YPD plates with G418 concentrations of 0.5 g/L, 1 g/L, 2 g/L, 3 g/L, 4 g/L, and 5 g/L separately, and cultivating the YPD plates, then screening to obtain the transformants; (4) fermentation of genetic recombinant human collagen inoculating the screened transformants to BMGY medium, and subjecting the inoculated BMGY medium to shaking cultivation for 24 hours, inoculating the cultivated BMGY medium as the first order seed to a fermentation tank loaded with FBS medium to cultivate at a pH of 5.0 for 16-20 hours, then inoculating the cultivated FBS medium as the second order seed to a fermentation tank loaded with FBS medium to perform an induction fermentation for 36-42 hours, with a growth temperature of 30 °C, an inducing temperature lower than the growth temperature, a pH of 5.0, and a dissolve oxygen in a range of 20%-30%; (5) purification of genetic recombinant human collagen subjecting the fermentation broth in step (4) to solid-liquid separation to give supemate; subjecting the supernate to microfiltration by a hollow fiber microfiltration system with a pore diameter of 0.1 pm or 0.22 pm, collecting the filtrate; subjecting the filtrate to desalination and concentration by ultrafiltration with a hollow fiber ultrafiltration system with a molecular weight cutoff of 10 KD, collecting the concentrated solution; then subjecting the concentrated solution to ion-exchange column chromatography with CM Sepharose FF to obtain the genetic recombinant human collagen with a purity of 95% or more.
In one embodiment, the present invention also provides a gene encoding a recombinant human collagen, the gene is one of the following nucleotide sequences: 1) SEQ ID No: 2 in the sequence list; 2) polynucleotides encoding the protein sequence of SEQ ID No: 1 in the sequence list; 3) DNA sequence, which has a homology of 90% or more to the DNA sequence defined by SEQ ID No: 2 in the sequence list and encodes the protein with the same functions as that of the protein encoded by SEQ ID No: 2; 4) DNA sequence encoding the protein derived from SEQ ID No: 1, the protein derived from SEQ ID No: 1 is a polypeptide having the same activity as that of SEQ ID No: 1, obtained by addition or deletion of 15 or less amino acid residues at N-terminal of the amino acid sequence of SEQ ID No: 1; 5) DNA sequence encoding the protein derived from SEQ ID No: 1, the protein derived from SEQ ID No: 1 is a polypeptide having the same activity as that of SEQ ID No: 1, obtained by addition or deletion of 15 or less amino acid residues at C-terminal of the amino acid sequence of SEQ ID No: 1; 6) DNA sequence coding the protein derived from SEQ ID No: 1, the protein derived from SEQ ID No: 1 is a polypeptide having the same activity as that of SEQ ID No: 1, obtained by substitution, deletion or addition of one or more amino acid residues in the amino acid sequence of SEQ ID No: 1.
For the above-described gene encoding collagen, similarly, the gene which can encode the collagen having the similar sequence as that of said collagen and has the similar activities as that of said collagen, may come into the protection scope of the present invention.
In one preferable embodiment, the gene is SEQ ID No: 2 in the sequence list.
In one embodiment, the present invention also provides an expression vector containing said gene; and a cell line containing said gene.
In one embodiment, the present invention is achieved by the following technical solution. Gly-X-Y repeating sequence of human collagen type I, preferably hydrophilic Gly-X-Y, is used as the minimum repeating unit to be permuted and combined. At the same time, in order to facilitate the post-purification, the ratio of the basic amino acid is adjusted, the isoelectric point is designed to about 9.5, and the collagen comprises 399 amino acids, the sequences of which are shown as follows, with reference to SEQ ID No: 1 in the sequence listing:
Gly Pro Pro Gly Ser Pro Gly Pro Lys Gly Pro Gin Gly Glu Lys 15
Gly Ser Pro Gly Pro Ala Gly Pro Lys Gly Ser Pro Gly Ser Gin 30
Gly Lys Pro Gly Asn Asp Gly Asn Pro Gly Glu Lys Gly Ser Gin 45
Gly Ser Pro Gly Glu Lys Gly Ser Pro Gly Lys Pro Gly Ser Pro 60
Gly Pro Lys Gly Lys Asp Gly Lys Pro Gly Pro Ala Gly Ser Pro 75
Gly Glu Lys Gly Ser Gin Gly Asn Asp Gly Ser Pro Gly Pro Lys 90
Gly Asn Pro Gly Glu Pro Gly Lys Pro Gly Glu Lys Gly Asn Pro 105
Gly Pro Pro Gly Asn Asp Gly Pro Ala Gly Lys Pro Gly Ser Gin 120
Gly Ser Gin Gly Glu Pro Gly Pro Asn Gly Pro Gin Gly Glu Lys 135
Gly Glu Gin Gly Asn Pro Gly Ser Pro Gly Glu Gin Gly Glu Pro 150
Gly Pro Gin Gly Ser Pro Gly Glu Pro Gly Lys Pro Gly Ser Gin 165
Gly Lys Pro Gly Ser Pro Gly Asn Pro Gly Ser Pro Gly Asp Lys 180
Gly Glu Lys Gly Glu Pro Gly Glu Lys Gly Ala Arg Gly Asn Pro 195
Gly Pro Gin Gly Pro Lys Gly Ala Asn Gly Gin Pro Gly Asn Pro 210
Gly Glu Lys Gly Ser Gin Gly Glu Pro Gly Asn Pro Gly Ser Gin 225
Gly Asn Pro Gly Asn Pro Gly Glu Pro Gly Glu Lys Gly Glu Lys 240
Gly Ala Arg Gly Pro Lys Gly Asp Lys Gly Asn Pro Gly Pro Lys 255
Gly Ala Asp Gly Ser Pro Gly Lys Asp Gly Lys Asn Gly Glu Pro 270
Gly Ser Pro Gly Lys Pro Gly Pro Gin Gly Glu Pro Gly Asp Lys 285
Gly Asn Asp Gly Pro Gin Gly Pro Ala Gly Ser Pro Gly Glu Lys 300
Gly Glu Pro Gly Asp Lys Gly Ala Arg Gly Gin Pro Gly Ser Pro 315
Gly Asn Gin Gly Ser Gin Gly Asn Asp Gly Gin Pro Gly Pro Lys 330
Gly Glu Pro Gly Asn Asp Gly Glu Lys Gly Ser Gin Gly Pro Pro 345
Gly Ser Pro Gly Pro Gin Gly Asn Pro Gly Pro Gin Gly Asn Asp 360
Gly Ala Arg Gly Glu Lys Gly Glu Lys Gly Ser Pro Gly Ser Pro 375
Gly Asn Asp Gly Asn Pro Gly Ala Arg Gly Lys Pro Gly Ser Pro 390
Gly Pro Lys Gly Asn Gin Gly Lys Pro 399
The above-described designed amino acid sequences are analyzed according to the present invention, and the corresponding nucleotide sequences are designed based on the codon bias of Pichia pastoris, shown as follows, with reference to SEQ ED No: 2 in the sequence listing: 1 ggtccaccag gttctccagg tccaaagggt ccacaaggtg aaaagggttc tccaggtcca 61 gctggtccaa agggttctcc aggttctcaa ggtaagccag gtaacgacgg taacccaggt 121 gaaaagggtt ctcaaggttc tccaggtgaa aagggttctc caggtaagcc aggttctcca 181 ggtccaaagg gtaaggacgg taagccaggt ccagctggtt ctccaggtga aaagggttct 241 caaggtaacg acggttctcc aggtccaaag ggtaacccag gtgaaccagg taagccaggt 301 gaaaagggta acccaggtcc accaggtaac gacggtccag ctggtaagcc aggttctcaa 361 ggttctcaag gtgaaccagg tccaaacggt ccacaaggtg aaaagggtga acaaggtaac 421 ccaggttctc caggtgaaca aggtgaacca ggtccacaag gttctccagg tgaaccaggt 481 aagccaggtt ctcaaggtaa gccaggttct ccaggtaacc caggttctcc aggtgacaag 541 ggtgaaaagg gtgaaccagg tgaaaagggt gctagaggta acccaggtcc acaaggtcca 601 aagggtgcta acggtcaacc aggtaaccca ggtgaaaagg gttctcaagg tgaaccaggt 661 aacccaggtt ctcaaggtaa cccaggtaac ccaggtgaac caggtgaaaa gggtgaaaag 721 ggtgctagag gtccaaaggg tgacaagggt aacccaggtc caaagggtgc tgacggttct 781 ccaggtaagg acggtaagaa cggtgaacca ggttctccag gtaagccagg tccacaaggt 841 gaaccaggtg acaagggtaa cgacggtcca caaggtccag ctggttctcc aggtgaaaag 901 ggtgaaccag gtgacaaggg tgctagaggt caaccaggtt ctccaggtaa ccaaggttct 961 caaggtaacg acggtcaacc aggtccaaag ggtgaaccag gtaacgacgg tgaaaagggt 1021 tctcaaggtc caccaggttc tccaggtcca caaggtaacc caggtccaca aggtaacgac 1081 ggtgctagag gtgaaaaggg tgaaaagggt tctccaggtt ctccaggtaa cgacggtaac 1141 ccaggtgcta gaggtaagcc aggttctcca ggtccaaagg gtaaccaagg taagcca
The preparation method of the above-described recombinant human collagen comprises the following steps: 1. Constitution of the expression vector of the genetic recombinant human collagen Based on the designed amide acid sequences of the genetic recombinant human collagen, designing and synthesizing the amide acid sequences of the genetic recombinant human collagen by artificial total gene synthesis in accordance with the codon bias of Pichia. Pastoris, meanwhile, adding the cleavage site CTCGAG of the Xho I endonuclease and the signal peptide cleavage site sequence AAAAGA of Pichia. Pastoris at 5' end, and adding the cleavage site GAATTC of the EcoR I endonucleas at 3' end. The designed sequences are synthesized by Shanghai Sangon biotechnology engineering Co., LTD, referred to as NCOL, and the sequences are shown as follows, with reference to SEQ ID No: 3 in the sequences listing: 1 ctcgagaaaa gaggtccacc aggttctcca ggtccaaagg gtccacaagg tgaaaagggt 61 tctccaggtc cagctggtcc aaagggttct ccaggttctc aaggtaagcc aggtaacgac 121 ggtaacccag gtgaaaaggg ttctcaaggt tctccaggtg aaaagggttc tccaggtaag 181 ccaggttctc caggtccaaa gggtaaggac ggtaagccag gtccagctgg ttctccaggt 241 gaaaagggtt ctcaaggtaa cgacggttct ccaggtccaa agggtaaccc aggtgaacca 301 ggtaagccag gtgaaaaggg taacccaggt ccaccaggta acgacggtcc agctggtaag 361 ccaggttctc aaggttctca aggtgaacca ggtccaaacg gtccacaagg tgaaaagggt 421 gaacaaggta acccaggttc tccaggtgaa caaggtgaac caggtccaca aggttctcca 481 ggtgaaccag gtaagccagg ttctcaaggt aagccaggtt ctccaggtaa cccaggttct 541 ccaggtgaca agggtgaaaa gggtgaacca ggtgaaaagg gtgctagagg taacccaggt 601 ccacaaggtc caaagggtgc taacggtcaa ccaggtaacc caggtgaaaa gggttctcaa 661 ggtgaaccag gtaacccagg ttctcaaggt aacccaggta acccaggtga accaggtgaa 721 aagggtgaaa agggtgctag aggtccaaag ggtgacaagg gtaacccagg tccaaagggt 781 gctgacggtt ctccaggtaa ggacggtaag aacggtgaac caggttctcc aggtaagcca 841 ggtccacaag gtgaaccagg tgacaagggt aacgacggtc cacaaggtcc agctggttct 901 ccaggtgaaa agggtgaacc aggtgacaag ggtgctagag gtcaaccagg ttctccaggt 961 aaccaaggtt ctcaaggtaa cgacggtcaa ccaggtccaa agggtgaacc aggtaacgac 1021 ggtgaaaagg gttctcaagg tccaccaggt tctccaggtc cacaaggtaa cccaggtcca 1081 caaggtaacg acggtgctag aggtgaaaag ggtgaaaagg gttctccagg ttctccaggt 1141 aacgacggta acccaggtgc tagaggtaag ccaggttctc caggtccaaa gggtaaccaa 1201 ggtaagccat aagaattc subjecting pPIC9K and the human collagen synthesized by artificial total gene synthesis to double digestion with enzymes Xho I and EcoR I, recovering the enzyme-digested product, ligating the product by using DNA ligase, transforming the ligation product into Colon bacillus, and extracting the plasmids referred to as pPIC9K-NCOL; 2. Electrotransformation of Pichia pastoris mixing 10 pg pPIC9K-NCOL plasmid linearized by Sal I endonuclease with 80 μΐ Pichia pastoris competent cells, transferring the mixture into a 0.2 cm ice-precooled electroporation cup, subjecting the mixture to electrical shock for 4-10 milliseconds, adding 1 ml ice-precooled 1 mol/L sorbitol solution thereto and mixing, spreading the resulting mixture on a MD medium plate, then culturing inverted MD medium plate at 30°C for 2-3 days, such that bacterial colonies grow on the MD medium plate; 3. Screening the multi-copy inserted recombinant correspondingly inoculating the bacterial colonies on the MD medium plate with sterile toothpicks to YPD plates with G418 concentrations of 0.5 g/L, 1 g/L, 2 g/L, 3 g/L, 4 g/L, and 5 g/L separately, cultivating the YPD plates at 30°C, then screening to obtain the transformants; 4. Fermentation of genetic recombinant human collagen inoculating the screened transformants to 400 ml BMGY medium, and subjecting the inoculated BMGY medium to shaking cultivation at 30°C for 24 hours, inoculating the cultivated BMGY medium as the first order seed to a 5 L fermentation tank loaded with 4L FBS medium to cultivate at a temperature of 30°C and a pH of 5.0 for 16-20 hours, then inoculating the cultivated FBS medium as the second order seed to a 150 L fermentation tank loaded with FBS medium to perform an induction fermentation for 36-42 hours, with a growth temperature of 30°C, an inducing temperature of 29°C, a pH of 5.0, a dissolve oxygen in a range of 20%-30%; the fermentation expression quantity is 10 g/1 or more; 5. Purification of genetic recombinant human collagen subjecting the fermentation broth to solid-liquid separation to give supemate; subjecting the supernate to microfiltration by a hollow fiber microfiltration system with a pore diameter of 0.1 pm or 0.22 pm, collecting the filtrate; subjecting the filtrate to desalination and concentration by ultrafiltration with a hollow fiber ultrafiltration system with a molecular weight cutoff of 10 KD, collecting the concentrated solution; then subjecting the concentrated solution to ion-exchange column chromatography with CM Sepharose FF to obtain the genetic recombinant human collagen with a purity of 95% or more.
The step 5 of purification of genetic recombinant human collagen according to the present application comprises the following steps: subjecting the fermentation broth to solid-liquid separation to give supemate; subjecting the supernate to microfiltration by a hollow fiber microfiltration system with a pore diameter of 0.22 pm, collecting the filtrate; subjecting the filtrate to desalination and concentration by ultrafiltration with a hollow fiber ultrafiltration system with a molecular weight cutoff of 10 KD, collecting the concentrated solution; then subjecting the concentrated solution to ion-exchange column chromatography with CM Sepharose FF to obtain the genetic recombinant human collagen with a purity of 95% or more.
The human collagen according to the present application has the characters of high expression quantity, excellent water solubility and high purity, which lay the foundation for the application of the genetic recombinant human- source collagen.
Example
Hereafter, the detection methods and the material sources in the examples according to the present application are described. pPIC9K is available from Invitrogen Corporation.
Xho I and EcoR I are available from Thermo Fisher Corporation.
Example 1 1. Constitution of the expression vector of the genetic recombinant human collagen 1.1 Obtaining of gene
Based on the designed amide acid sequences of the genetic recombinant human collagen, the amide acid sequences of the genetic recombinant human collagen by artificial total gene synthesis were designed and synthesized in accordance with the codon bias of Pichia. Pastoris. Meanwhile, the cleavage site CTCGAG of the Xho I endonuclease and the signal peptide cleavage site sequence AAAAGA of Pichia. Pastoris were added at 5' end, and the cleavage site GAATTC of the EcoR I endonucleas were added at 3' end. The designed sequences were synthesized by Shanghai Sangon biotechnology engineering Co., LTD, referred to as NCOL, and the sequences refer to SEQ ID No: 3 in the sequences listing:
1.2 Double digestion of pPIC9K vector and NCOL
The pPIC9K vector and the human collagen synthesized by artificial total gene synthesis were subjected to double digestion with enzymes Xho I and EcoR I. wherein, pPIC9K is available from Invitrogen Corporation, and the double enzymes system is shown as follows:
Total system 200 pL
Total system 200 pL
After addition of the above reagents, they were mixed uniformly. The resulting mixture was digested in 37°C water bath for 2 hours. The specific bands were recovered from agarose gel with mass percentage concentration of 1.5% by using DNA purification kits, according to product specifications.
1.3 Ligation of the pPIC9K vector and NCOL pPIC9K recovered after digestion and NCOL were ligated by using T4 DNA ligase, and the reaction system is shown as follows:
Total system 20 pL
The above reagents were mixed uniformly, and subsequently the reaction was performed at 16°C for 12 hours. 1.4 Transformation of the ligation products A tube containing 100 μΐ of frozen DH5a competent cells was thawed on ice, then the above ligation products were added thereto. The tube was flicked softly by fingers to mix uniformly, then placed in ice for 30 minutes, subjected to heat shock in water bath at 42°C for 90 seconds. The tube was quickly transferred to ice bath, so that the cells were cooled for 2 to 3 minutes. 800 μΐ antibiotic-free LB fluid medium was added thereto and mixed, then incubated at 37°C for 50 minutes. 100-200 μΐ of bacteria solution was drawn and spreaded on a solid LB plate with kanamycin (50 pg/ml). The solid LB plate was inverted in an incubator at 37°C and incubated for 12 to 18 hours. The single colony was selected and inoculated into a liquid LB medium containing ampicillin (100 pg/ml), then subjected to shake cultivation at 37°C overnight. The plasmid was extracted with a plasmid extraction kit according to instructions and sequenced. The plasmid was referred to as pPIC9K-NCOL, and the plasmid map is shown in Figure 1. 2. Electrotransformation of Pichia pastoris
The pPIC9K-NCOL plasmid was firstly linearized by Sal I endonuclease, and the reaction system was shown as follows:
Total system 1600 μΐ
Following the addition and mixing of the above reagents, they were reacted in water bath at 37°C for 5 hours. Before reaction terminal, 5 μΐ of the mixture was taken to detect whether the enzyme digestion was completed by agarose gel electrophoresis with a mass percent concentration of 1.0%. Subsequently, the linearized plasmid was recovered by using a DNA purification kit. 10 pg of the pPIC9K-NCOL plasmid linearized by Sal I endonuclease was mixed with 80 μΐ Pichia pastoris GS115 competent cells, the mixture was transferred into a 0.2 cm ice-precooled electroporation cup, then subjected to electrical shock for 4-10 milliseconds at a voltage of 1.5 kV, a capacitance of 25 pF, and a resistance of 200 Ω. 1 ml of ice-precooled 1 mol/L sorbitol solution was added thereto and mixed, then the resulting mixture was spreaded on a MD medium plate. The MD medium plate was cultivated invertedly at 30°C for 2-3 days, such that bacterial colonies grew on the MD medium plate. 3. Screening the multi-copy inserted recombinant
The bacterial colonies grew on the MD medium plate were correspondingly inoculated with sterile toothpicks to YPD plates with G418 of 0.5 g/L, 1 g/L, 2 g/L, 3 g/L, 4 g/L, and 5 g/L, respectively. The bacterial colonies on YPD plates were cultivated at 30°C, then screened to obtain the transformants. 4. Fermentation of genetic recombinant human collagen
The screened transformants were inoculated to 400 ml BMGY medium, subjected to shaking cultivation at 30°C for 24 hours, then inoculated as the first order seed to a 5 L fermentation tank loaded with 4L FBS medium (containing glycerol 40g, K2SO4 18.2g, H3PO4 26.7 ml, CaSC>4 ·2Η2θ 0.93g, MgSC>4 14.9g, and KOH 4.13g in 1L) to cultivate at a temperature of 30°C and a pH of 5.0 for 16-20 hours. Then, the cultivated FBS medium was inoculated as the second order seed to a 150 L fermentation tank loaded with 80L FBS medium to perform induction fermentation. During the fermentation, the growth temperature was 30°C, the inducing temperature was 29°C, the pH was 5.5, and the dissolve oxygen was in a range of 20%-30%. The sharply raised dissolved oxygen indicated that glycerol in the basal salt medium was exhausted. Glycerol with a mass percentage concentration of 50% was added thereto at a fed-batch rate of 18.15ml/h/L, wherein the glycerol contained 12 ml/L of trace elements PTM1 (containing CuSC>4 · 5H20 6g, Nal 0.08g, MnS04.H20 3g, Na2Mo04.H20 0.2g, H3BO3 0.02g, H2SO4 5ml, C0CI2.6H2O 0.5g, ZnCb 20g, FeS04.7H20 75g , and biotin 0.2g in 1L). The dissolved oxygen was maintained at 20% to 30%. The feeding was stopped when the wet bacterial weight of fermentation broth reached 180 mg/ml. Glycerol was exhausted after one hour hunger. 20 L of methanol with a mass percentage concentration of 75% was added by fed-batch at a rate of 7.5ml/L/h for 2 to 3 hours to induce fermentation, wherein the methanol contained 12 ml/L of PTM1 trace elements. Then, the rate was increased to 10.9 ml/L/h for fermentation. By adjusting the rotation rate, the tank pressure and the ventilation, the dissolved oxygen was greater than 20%. The induction fermentation was carried out for 36 to 42 hours. The electrophoretogram of the fermentation supernatant was shown in Figure 2. The expression quantity was up to lOg /L or more. 5. Purification of genetic recombinant human collagen 5.1 Solid-liquid separation and decolorization of the fermentation broth
After fermentation, the fermentation broth was centrifuged. 80L of supernatant was subjected to solid-liquid separation by a hollow fiber microfiltration system with a pore diameter of 0.1 pm to give filtrate. Then, the filtrate was subjected to desalination and concentration by ultrafiltration with a hollow fiber ultrafiltration system with a molecular weight cutoff of 10 KD to give a concentrated solution. A large number of yellow-green substances obtained during fermenting Pichia. Pastoris were removed by the above two-step ultrafiltration. 5.2 Ion-exchange chromatography of the recombinant human growth hormone
The above concentrated solution was subjected to anion exchange chromatography, wherein the filler was CM Sepharose FF purchased from GE Corporation. 3-fold column volume of lOmmol/L citrate buffer with pH of 5.0 was used to balance the column. Samples were loaded. 10-fold column volume of lOmmol/L citrate buffer (containing 0.5 mol/L NaCl) with pH of 5.0 was used to elute, and the eluent was collected. The eluent was subjected to ultrafiltration and desalination by a roll ultrafiltration membrane with a molecular weight of 10KD. The concentrated solution was collected and freeze-dried by a freezer dryer to give the genetic recombinant human collagen with a purity of 95% or more. The purification results were shown in Figure 2. 6. Cytotoxicity and cell proliferation- promoting assay of the genetic recombinant human collagen
The cultivated fibroblasts L929 were diluted to 6xl03/ml of single cell suspension with DMEM/F12 culture solution containing 10% of FBS. ΙΟΟμΙ of cell suspension was inoculated to three 96-well plates, 40 wells per plate. The plates were placed in an incubator containing 5% (volume fraction) of CO2 to perform cultivation at 37°C for 24 hours. The original culture solution was discarded. 100 pi of experimental group (genetic recombinant human collagen, bovine collagen) lOmg/ml prepared solutions and the negative control solution (pure cell culture solution) were added to each well. Each group included 8 wells. The three plates were transferred to the incubator containing 5% (volume fraction) of CO2. After inoculation 2, 4, 7 days, one plate was taken out from each group. Each well was added with 50 μΐ of MTT (thiazolyl blue), then cultivated at 37°C for another 2 hours. The original culture solution was discarded, then 150 μΐ of dimethyl sulfoxide was added immediately into each well. The plates were replaced at room temperature and gently shaken for 10 to 15min. The absorbance of every well was measured on a microplate reader at the wavelength of 490 nm. The relative proliferation rate (RGR) was calculated as follows: RGR (%) = (absorbance of the experimental group / absorbance of the negative control group) χ 100%. It can be seen from Figures 3 and 4 that the survival rates of the genetic recombinant human collagen and the bovine collagen were more than 100%, and increased with the increased days of cell cultivation; and the genetic recombinant human collagen could effectively promote the proliferation of fibroblasts, and the data of genetic recombinant human collagen were slightly higher than the bovine collagen. In conclusion, the genetic recombinant human collagen has a cytotoxicity of 0, and it has good biocompatibility.
Example 2
The experimental procedures of Example 2 were the same as that of Example 1, except that the hollow fiber microfiltration system with a pore diameter of 0.1 pm in the purity step 5 was replaced by the hollow fiber microfiltration system with a pore diameter of 0.22 pm. The product in Example 2 is genetic recombinant human collagen with a purity of 95% or more, and it has a cytotoxicity of 0 and good biocompatibility.
Example 3
According to hydrophilic Gly-X-Y of human collagen, it was designed that 15 amino acid sequences were added to the genetic recombinant human collagen at N-terminal of the amino acid. The 15 amino acid sequences were GPAGSQGPPGEKGND. The genetic recombinant human collagen sample prepared by the same method as that of Example 1 has a purity of 95% or more and good biocompatibility.
Example 4
According to hydrophilic Gly-X-Y of human collagen, it was designed that 15 amino acid sequences were added to the genetic recombinant human collagen at C-terminal of the amino acid. The 15 amino acid sequences were GEPGQPGARGSPGSQ. The genetic recombinant human collagen sample prepared by the same method as that of Example 1 has a purity of 95% or more and good biocompatibility.
From the experimental data of the above examples, it can be seen that the genetic recombinant human collagen designed and prepared according to the present invention has a purity of 95% or more and good biocompatibility.

Claims (11)

  1. What h claimed Is:
    1, A fecombmani human collagen, characterized in -that, the eollapn comprises om <a* more of the following family members: 1} protein Imvkg the ammo aeM sequence 0FSBQ ID Mo: 1 in the sequence list:
  2. 2) . SEQ ID Np; 1 derived protein, having a sequen.ee homology of 90% or more to the amino acid sequence of SEQ ID Mo: 1 and having the same activity as that of SBQ ID Me: i; 3) SEQ ID Mo: J derived, protein, having the same activity as that of SEQ ID Mo; i, obtained by addition or deletion of 1$ or less amino acid residues at MTerinlnal of the amino acid sequence of SEQ ID Mb: 1; 4) SEQ ID No: I derived protein, having the same activity as that of SEQ ID No: E obtained by addition or deletion of 15 or less amino acid residues at CMtermina! of the amino acid sequence of SEQ ID No: 1; 5) SEQ ID No; I derived protein, having the same activity as that of SEQ: ID Mo; t, obtained by substitution, deletion or addition of one or more amino acid residues in the amino acid residue sequence of SEQ ID No: L
    2, The recombinant human collagen of claim 1, characterized in that, the protein is SEQ ID Mb; I in the sequence list
  3. 3, The recombinant human collagen of claim I or % chameierized in that the isoelectric point of the protein Is 9,5.
  4. 4, The recombinant human collagen of any one of claims S-3, characterized In that, the host cell lor expressing theprotem is Piekia, Pmi&amp;rm
  5. 5, A preparation method for the recombinant human collagen of any one of claims M, characterized In that, the method comprises the following steps: fl) preparation of plasmids: synthesizing the sequence of SEQ ID No: 3 In the sequence list by arilBesal total gene synthesis, subjecting the pFIC9K vector arid the human collagen of SEQ ID No:3 synthesized by artificial total gene synthesis to double digestion with enzymes Xho! and EeoR t recovering the enzyme-digested product, ligating the product-by using DNAIlgase, transforming the ilgation product into Cofen bmilim, and extracting plasmids; (2) transformation:; mixing the prepared plasmids in step (1) with Piekw paMims for transformation, and obtaining transformed bacterial colonies; (3) screening the multi-copy inserted recombinant: cultivating the transformed bacterial colonies obtained in step (2), and screening transformants; (4) fermentation; Inoculating and fermenting foe screened transformants in step (3) to obtain fermentation broth; (5) purification: successively subjecting the fermentation broth in step (4) &amp;> solid-liquid separation, filtration, concentration. and ion-exchange column chromatography to obtain the product, fi, lie preparation method of claim 5, characterized in that, the purification in step (5) comprises the steps of; .subjecting the fermentation broth obtained in step (4) to solid-liquid separation to give supernate; subjecting the supernate to mierofilfeatidn by a hollow fiber ntleroiftratinn system with a pore diameter of ¢1.22 pm, coiiectmg ihefiltrate;: subjecting the filtrate fo desalination and concentration by uhraffitration with a hollow fiber ultrafijtration system, with a molecular weight cutoff of 10. KB;, collecting the concentrated solution; then subjecting the concentrated solution: to ion-exchange column chromatography with CM Sepharose FF to obtain the product,·
  6. 7, The preparation method of claim 5 or 6, characterized in that, the method compri ses the following steps;: (1) prepared ο» of plasmids synthesizing foe sequence of SBQ ID No; 3 in the sequence list by artificial total gene synthesis, subjecting, the pPIC9K vector and the human collagen of SEQ ID No :3 synthesized hy artificial: total gene synthesis to: double digestion with enzymes Xho 1 and EeoR:!, recovering the enzyme-digested product, ligating the product by using DNA ligase, transforming foe ligation product into Cohn hm’iihis, and extracting the plasmids referred to as pnmK-mou (2) efectrpiransformation oFJ%fofe pastom mixing the pflC9K-MCOL plasmid linearized by Sal i" endonuclease with Pidiia pustoris competent cells» transferring the mixture into a iee-preeooled^ electroporation cup». sufefocUng the: mixture to electrical shock, for 4*10 milliseconds, adding iee-precpofod sorbitol solution thereto and mixing, spreading, the resulting mixture on a ME) medium plate, then. culturing inverted MD medium plate for 2-3 days, such that bacterial colonies grow on the MD medium plate: (3) screening multi-copy inserted recombinant correspondingly inoculating the bacterial colonies on the MD medium plate with sterile toothpicks to YPD plates with 04! δ concentrations of 0.5 g/L, 1 g/L, 2 g/L, 3 g/l ,, 4 and 5 g/L separate I ly; cultivating the YPD plates, then screening to obtain the transformants;;: ,(4) formerrtatioitpf genetic recombinant human collagen inoculating foe screened transformants fo BMC5Y medium, and subjecting; the 'inoculated BMGY tnediutn to shaking cultivation for 24 hours. Inoculating the cultivated BMCIY medium as the first order seed to a fermentation tank loaded with FBS medium to cultivate at a pH of'5,0 for 16-20 hours, then inoculating the cultivated FBB medium as the second order seed to a fermentation, lank loaded with PBS medium to perform an. induction fermentation for 36-42 hours, with a growth temperature of 30 ”C, ah inducing temperature lower than the growth temperature, a pH of 5,0» and a dissolve oxygen in a range of 20%-3(}%; (5} purification of genetic recombinant human collagen subieciing the formenimion broth in step (4) to solidrikpid separation to collect foe supernate; suhjeefing the supemate to mlcroflitration by a hoilbtv fiber microfiltration system with a pore diameter of 0,1 pm or 0,22 pm, -criileetiog the filtrate; subjecting the filtrate to desalination and ^concentration fey uitrafiltrafion with a hollow fiber oltrafidfoation system with a molecular weight cutoff of 10 KD„ collecting the concentrate solution; thee subjecting the concentrated solution to ion-exchange column chromatography with CM Sepbatose FF to obtain the genetic recombinant human collagen with a purity of 95% or more,
  7. 8, A gene encoding a recombinant human collagen, characterized in that, the gene is one of the fu I lowing nucleotide sequences: 1) SEQ J;D'19bt 2 in the. sequence list; 2) - polynucleotides encoding the protein sequence of SEQ ID No: 1 in the sequence list; 3) DMA sequence, which has a homology of 90% or more to the DMA sequence defined by .SEQ ID No: 2 m the sequence list and encodes the protein with the same iunetiorss as that of the protein encoded by SEQ ID No: 2; 4) I)NA sequence encoding the protein derived from SEQ ID No: 1,. the protein derived fern SEQ ID No; I is a polypeptide having the same activity as that of SEQ ID No: 1, obtained by addition or deletion, of 15 or less amino acid residues at N-terminal of the amino acid sequence of SEQ ID No: 1; Sf DMA sequence encoding the protein derived from SEQ ID. No: L the protein derived ffpnv SEQ ID Mo; 1 js a polypeptide having the same activity as that of SEQ: ID No: 1, obtained: by addition m deletion of 15 or less: .amino acid residues at C-terminal of the. amino ac id sequence of SEQ ID No: 1; d) DMA sequence coding the protein derived from SEQ ID No: I, the protein derived from, SEQ ID No; I Is a, polypeptide having the same activity as that of SEQ: ID 'No: 1., obtained by substitution* deletion or addition: of one or more amino acid residues in the amino acid sequence of SEQ ID No: 1.
  8. 9. The gene of claim 8* .characterized in that* the gene is SEQ ID No: 2 in the sequence list. Hi, An expression vector, containing the gene of cla im 8 or 9 ,
  9. 11, A cell line, containing the gene of claim 8 or 9.
  10. 12, A biological material for tissue engineering and shincare, characterized in that, the biological material contains the human collagen of claims 1-4, the recombinant human collagen -encoded by the gene of clai m 8 or 9,
  11. 13, The hitman collagen of claims 1-4, the recombinant human collagen prepared by the method of elatms: 5-7, the gene coding recombinant human collagen of claim 8 or 9, the purity is 95% or more.
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