CN117384276A - Recombinant collagen and preparation method and application thereof - Google Patents

Abstract

The invention relates to the technical field of genetic engineering, in particular to recombinant collagen, a preparation method and application thereof. The amino acid sequence of the recombinant collagen is shown as SEQ ID No.1 or SEQ ID No. 8. The recombinant collagen is expressed at a high level outside cells, the culture is easy to amplify, the target protein can be produced at a high yield by utilizing a high-density fermentation mode, the secretory expression is beneficial to the separation and purification of an expression product, the production cost is reduced, and the recombinant collagen has certain post-translational modification functions, such as glycosylation and the like, on the expressed exogenous protein.

Description

Recombinant collagen and preparation method and application thereof

Technical Field

The invention relates to the technical field of synthetic biology, in particular to recombinant collagen and a preparation method and application thereof.

Background

Collagen, which is an important natural biological protein, has unique functional characteristics such as good biocompatibility, bioactivity, degradability and the like, is widely applied to the fields of biology, cosmetology, medical material fields, food health care and the like, particularly plays a very important role in medical fields such as burn treatment, wound treatment, hemostasis, ophthalmic treatment and stomatology treatment, and simultaneously, has started to be used as a scaffold material for constructing organs abroad, but the problem of limiting the application of collagen to the greatest extent is the source of collagen.

Currently, collagen is obtained in two main ways, including traditional methods derived from animal tissues and heterologous protein expression. The traditional method from animal tissue mainly extracts natural collagen and collagen peptide from animal tissue and marine organisms (livestock and poultry bones, pig skin, sea cucumber, fish scales and fish skin), and has the problems of simple and convenient process: 1) The collagen extraction efficiency is low, the molecular weight of the extracted collagen is generally not high, and the extracted collagen contains more impurities; 2) The molecular weight of the extracted collagen is more than 500 and Da under the limit of an extraction process, and the small collagen is difficult to extract; 3) The different extraction processes, especially the acid method and the alkali method, can pollute the environment and can not completely achieve green extraction. The expression of heterologous protein is to use engineering host cell to express exogenous introduced gene, and the heterologous protein is obtained after transcription and translation of cytoplasm by engineering host cell. Heterologous protein expression has many advantages over traditional methods derived from animal tissue: 1) The product is safe and the production process is controllable. The raw materials are relatively clear, the components are clear, and the pollution risks such as virus infection and the like are reduced. 2) The product quality is stable, and the batch repeatability is good. The specific collagen molecule is expressed by adopting a genetic engineering technology, the components are single, the stability of the strain is good, the difference between production batches is small, and the quality of the product is stable. 3) Good biocompatibility and reduced unnecessary immune response. 4) The production period of collagen obtained by microbial fermentation is relatively short, and the production cost is relatively low. The engineering host cell mainly comprises animal cells, plant cells and microorganism cells, and the microorganism cells have the advantages of high yield, short production period, simple culture, low cost, easy acquisition of high-density fermentation and the like compared with other cells in engineering host cell selection.

The host bacteria for recombinant expression of human collagen at present mainly comprise pichia pastoris, saccharomyces cerevisiae, hansenula, escherichia coli, and the like. Pichia pastorisPichia pastoris) The expression system is a foreign protein expression system, has the advantages of simple operation, easy culture, high growth speed, high expression quantity, low cost and the like of a prokaryotic expression system, and also has the characteristics of modification of foreign proteins such as glycosylation, protein phosphorylation and the like which are not possessed by the prokaryotic expression system. Over 200 different proteins have achieved successful expression in pichia pastoris over the last 20 years, with many products having been widely used in clinical diagnostic treatments and scientific research. In the pichia pastoris expression system, the exogenous gene is not in an autonomously replicating expression vector, but is integrated on a yeast chromosome together with the expression vector through homologous recombination, and is replicated and inherited together with the chromosome, so that the phenomenon of losing the exogenous gene can not occur; the pichia pastoris has strong aerobic growth preference, can realize high-density culture of cells, and is beneficial to large-scale industrial production; the pichia pastoris can secrete and express exogenous proteins at a high level, the accumulation of fermentation products does not produce toxic or side effect on the exogenous proteins, and the pichia pastoris secretes few proteins into a culture medium, so that the pichia pastoris is convenient to purify. Pichia pastoris has high fermentation expression yield, simple culture medium, stable expression protein and easy preparationIn purification, the protein has the ability to post-modify, such as glycosylation.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, an object of the present invention is to provide a recombinant collagen, a preparation method and use thereof, which are used for solving the problems of the prior art.

To achieve the above and other related objects, the present invention provides a recombinant collagen comprising one or more of the following features:

1) The amino acid sequence of the recombinant collagen is shown as SEQ ID No.1 or SEQ ID No. 8;

2) The recombinant collagen is protein which is derived from 1) and has collagen activity and is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence in 1).

Preferably, the recombinant collagen is type III collagen.

The invention also provides an isolated nucleic acid molecule, said polynucleotide encoding the aforementioned recombinant collagen.

The invention also provides an expression vector comprising the aforementioned nucleic acid molecule and a plasmid backbone.

The invention also provides a host cell comprising the aforementioned nucleic acid molecule or the aforementioned expression vector.

The invention also provides a preparation method of the recombinant collagen, which comprises one or more of the following steps:

1) The aforementioned host cells under suitable culture conditions;

2) Inducing the expression of the target gene of the host cell in the step 1) to obtain recombinant collagen;

3) Harvesting host cells and/or culture medium containing recombinant collagen, and separating and purifying the recombinant collagen in the step 2).

The invention also provides the use of the recombinant collagen, the nucleic acid molecule, the expression vector or the host cell in the preparation of cosmetics, skin care products or tissue materials.

As described above, the recombinant collagen and the preparation method and application thereof have the following beneficial effects:

(1) The recombinant collagen gene simultaneously contains the characteristics of the natural human collagen gene, and the expressed protein has the excellent characteristics of the natural human collagen; (2) In terms of the molecular length of the human collagen, the recombinant human collagen of the natural human collagen with the molecular weight close to a larger molecular weight can be expressed and produced in further yeast expression research by preparing different repeated numbers of the gene recombinant plasmids in the same direction and in series; (3) The preparation method of the gene recombinant plasmid with different repetition numbers connected in series in the same direction realizes the serial connection of the target genes (recombinant human collagen genes) with any repetition number only through simple PCR and homologous recombination transformation and screening; (4) The pichia pastoris engineering bacteria capable of inducing expression can realize high-level expression of recombinant human collagen outside cells, the culture is easy to amplify, the target protein can be produced in a high-density fermentation mode, the secretory expression is favorable for separation and purification of an expression product, the production cost is reduced, and the recombinant human collagen has certain post-translational modification functions, such as glycosylation and the like, on the expressed exogenous protein.

Drawings

FIG. 1 shows the SDS-PAGE detection gel of the recombinant collagen of the present invention, line1: GS115-pPIC9K-C induction 96 h, line2: GS115-pPIC9K-C induction 24 h, line3: GS115-pPIC9K-E induction 96 h, line4: GS115-pPIC9K-E induction 24 h, line5: GS115-pPIC9K-B induction 96 h, line6: GS115-pPIC9K-B induction 24 h.

FIG. 2 shows PCR gel electrophoresis patterns of recombinant collagen monomers of the invention, wherein the PCR products are prepared by taking Line1: marker, line 2-Line 8: GS115-pPIC9K-B as templates.

FIG. 3 shows the SDS-PAGE detection gel of recombinant collagen of the present invention, line1: pPIC9K-6*B induced 96 h, line2: pPIC9K-5*B induced 96 h, line3: pPIC9K-7*B induced 96 h, line4: GS115-pPIC9K induced 96 h, and Line5: marker.

FIG. 4 shows a schematic structure of the recombinant plasmid pPIC9K-C+B+E of the present invention.

FIG. 5 shows a diagram of a recombinant collagen SDS-PAG detection gel of the present invention, line1: pPIC9K-C+B+E induction 96 h, line2: pPIC9K-C+B+E induction 24 h, line3: pPIC9K-B induction 96 h, and Line4: marker.

FIG. 6 shows the SDS-PAG assay gel for recombinant human collagen according to the invention, line1 pPIC9K- (C+B+E) 2 induction 96 h, line2 pPIC9K- (C+B+E) 2 induction 72 h, line3 pPIC9K (C+B+E) 2 induction 48 h, line4 pPIC9K- (C+B+E) 2 induction 24 h, line5 Marker.

FIG. 7 shows a liquid chromatogram of recombinant collagen according to the present invention.

FIG. 8 shows the results of the hydrophilic-hydrophobic analysis of the recombinant collagen sequences of the present invention and other collagen sequences.

FIG. 9 shows a composition of BMGY medium of the invention.

FIG. 10 shows a graph of BMMY medium composition of the present invention.

FIG. 11 shows an MD plate composition of the present invention.

FIG. 12 shows a YPD plate composition of the invention.

Detailed Description

The invention provides a recombinant collagen comprising one or more of the following features:

1) The amino acid sequence of the recombinant collagen is shown as SEQ ID No.1 or SEQ ID No. 8;

2) The recombinant collagen is protein which is derived from 1) and has collagen activity and is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence in 1).

In some embodiments, the recombinant collagen is type III collagen.

Further, the protein fragment described in 2) specifically refers to: a protein having the amino acid sequence shown in SEQ ID No.1 or SEQ ID No.8 and having the function of recombinant collagen shown in SEQ ID No.1 or SEQ ID No.8, wherein the amino acid sequence in 1) may have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID No.1 or SEQ ID No.8, obtained by substituting, deleting or adding one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5 or 1-3) amino acids to the amino acid sequence shown in SEQ ID No.1 or SEQ ID No.8, or adding one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5 or 1-3) amino acids to the N-terminal and/or C-terminal end.

In some embodiments, the recombinant collagen is formed by the tandem of one or more repeat units having the amino acid sequence shown in SEQ ID No.1 or SEQ ID No. 8. More specifically, the recombinant collagen consists of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 repeating units with the amino acid sequence shown in SEQ ID No.1 in series. Preferably, the recombinant collagen is formed by tandem connection of 1-6 repeating units with the amino acid sequence shown as SEQ ID No.1 or SEQ ID No. 8.

In some embodiments, the recombinant collagen is formed by tandem connection of 2-6 stretch of repeating units having the amino acid sequence shown in SEQ ID No.1 or SEQ ID No. 8.

In some embodiments, the recombinant collagen further comprises an amino acid sequence comprising a purification tag or a secretion tag. More specifically, the purification tag is selected from one or more of His tag, GST tag, MBP tag, SUMO tag, or NusA tag; alternatively, the secretion tag is selected from DsbA, pelB, ompA, tolB, malE, lpp, torA or HylA or hybrid secretion tags.

In some embodiments, the His tag, also known as a histidine tag, is a sequence of 2 to 20 histidine residues attached to collagen. The histidine tag comprises 2 to 20 histidine residues, 5 to 15 histidine residues, 5 to 18 histidine residues, 5 to 16 histidine residues, 5 to 15 histidine residues, 5 to 14 histidine residues, 5 to 13 histidine residues, 5 to 12 histidine residues, 5 to 11 histidine residues, 5 to 10 histidine residues, 6 to 12 histidine residues, 6 to 11 histidine residues or 7 to 10 histidine residues. Histidine tags can be used to purify proteins by chromatographic methods using nickel-based chromatographic media.

In some embodiments, the recombinant collagen may further comprise green fluorescent protein GFP or red fluorescent protein RFP.

In some embodiments, the recombinant collagen further comprises a protease cleavage site. Protease cleavage sites can be used to cleave recombinantly produced collagen to remove portions of the polypeptide. The portion of the polypeptide that can be removed includes a secretion tag, a histidine tag, a fluorescent protein tag, and/or a beta-lactamase. Proteases include endo-, exo-, serine-, cysteine-, threonine-, aspartic-, glutamic-, and metallo-proteases. Exemplary protease cleavage sites include amino acids cleaved by thrombin, TEV protease, factor Xa, enteropeptidase, and rhinovirus 3C protease.

The invention also provides an isolated nucleic acid molecule encoding the aforementioned recombinant collagen.

In some embodiments, the nucleotide sequence of the nucleic acid molecule is set forth in SEQ ID No.2 or SEQ ID No. 9.

In some embodiments, the nucleic acid molecule is formed by concatenating one or more nucleic acid molecules having the nucleotide sequence set forth in SEQ ID No.2 or SEQ ID No. 9. More specifically, the nucleic acid construct is formed by tandem connection of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleic acid molecules having the nucleotide sequence shown in SEQ ID No.2 or SEQ ID No. 9. Preferably, the polynucleotides are formed by concatenating polynucleotides having 1-6 nucleotide sequences as shown in SEQ ID No.2 or SEQ ID No. 9.

As used herein, a "nucleic acid molecule" refers to a polynucleotide formed by the polymerization of deoxyribonucleotides or ribonucleotides.

The invention also provides an expression vector comprising the aforementioned nucleic acid molecule and a plasmid backbone.

In some embodiments, the backbone plasmid is selected from one or more of a pET expression vector, a pCW expression vector, a pUC expression vector, a pAO815 expression vector, a pPIC9K expression vector, a ppic3.5K expression vector, a ppiczαa expression vector, a ppiczαb expression vector, a ppiczαc expression vector, a pgapzαa expression vector, a pgapzαb expression vector, a pgapzαc expression vector, a pPICZ a expression vector, a pPICZ B expression vector, a pPICZ C expression vector, a pGAPZ a expression vector, a pGAPZ B expression vector, or a pGAPZ C expression vector. Preferably, the backbone plasmid is selected from the group consisting of a pPIC9K expression vector, a ppiczα expression vector or a pgapzαc expression vector.

The plasmid skeleton in the application is a circular DNA molecule or linear DNA molecule, and can autonomously replicate and express the inserted target gene in cells. The backbone plasmid may contain regulatory sequences such as promoters, replicons, enhancers, transcription and translation initiation and termination codons. The plasmid backbone is typically linked to the gene of interest to form an intact expression vector that can express a particular output in the cell.

The invention also provides a host cell comprising the aforementioned nucleic acid molecule or the aforementioned expression vector.

In some embodiments, the host cell is selected from a bacterial cell, a fungal cell, a plant cell, or an animal cell. More specifically, the bacterial cell is selected from the group consisting of E.coli, lactobacillus, bacillus licheniformis, bacillus subtilis, and Streptomyces; or, the fungal cell is selected from a mold cell or a yeast cell; or, the plant cell is selected from a tobacco cell or an arabidopsis cell; or, the animal cell is selected from a mammalian cell or an insect cell. Preferably, the yeast cell is selected from Saccharomyces cerevisiae, candida, torulopsis, rhodotorula, hansenula, pichia or Kluyveromyces; or, the insect cell is an Sf9 cell; or, the mammalian cell is selected from the group consisting of CHO cells, NS0 cells, sp2/0 cells, HEK293 cells, HKB11 cells, per.c6 cells, heLa cells, or CAP cells. More preferably, the host cell is pichia pastoris.

The invention also provides a preparation method of the recombinant collagen, which comprises one or more of the following steps:

1) Culturing the aforementioned host cell under suitable culture conditions;

2) Inducing the expression of the target gene of the host cell in step 1) to obtain recombinant collagen;

3) Harvesting host cells and/or culture medium containing recombinant collagen, and separating and purifying the recombinant collagen in the step 2).

In some embodiments, the culture conditions in step 1) comprise one or more of the following features:

1) The temperature of the culture is 20-35 ℃; more specifically, the temperature of the culture is 20-22 ℃, 22-25 ℃, 25-27 ℃, 27-30 ℃, 30-33 ℃ or 33-35 ℃; preferably, the temperature of the culture is 25-30 ℃;

2) The pH of the culture is 3-8; more specifically, the pH of the culture is 3-4, 4-5, 5-6, 6-7 or 7-8; preferably, the pH of the culture is 5-6;

3) The culture medium comprises one or more of the following components: phosphate, calcium salt, potassium salt, magnesium salt, inorganic base, glycerin and trace elements.

In some embodiments, the process induced in step 2) comprises one or more of the following features:

1) Substances that induce expression of genes of interest in host cells include methanol;

2) The induction time is 72-144 h; more specifically, the induction time is 72-84 h, 84-96 h, 96-108 h, 108-120 h, 120-132 h, or 132-144 h; preferably, the time of induction is 96-120 h.

In some embodiments, the process of step 3) isolation, purification comprises one or more of the following features:

1) The temperature for separation and purification is 0-15 ℃; more specifically, the separation and purification temperature is 0-2deg.C, 2-4deg.C, 4-6deg.C, 6-8deg.C, 8-10deg.C, 10-12deg.C or 12-15deg.C; preferably, the temperature of the separation and purification is 4-10 ℃;

2) The separation and purification process comprises ultrafiltration membrane concentration;

3) The separation and purification process comprises a chromatography process; more specifically, the chromatographic process is selected from adsorption chromatography, partition chromatography, affinity chromatography, gel chromatography or ion exchange chromatography.

The present invention provides the use of the recombinant collagen, the nucleic acid molecule, the expression vector or the host cell described above for the preparation of a cosmetic, a skin care product or a tissue material.

In some embodiments, the skin care product is a personal care product. In particular, the personal care product is selected from the group consisting of a facial mask, a skin cleanser such as soap, cleansing cream, cleansing emulsion, facial puff, facial cleanser, shampoo, conditioner or body wash.

In some embodiments, the tissue material is selected from a hydrogel or a collagen sponge.

The present invention also provides a composition comprising the aforementioned recombinant collagen and at least one additional ingredient, including a topical carrier or preservative.

In some embodiments, the topical carrier comprises a topical carrier selected from the group consisting of liposomes, biodegradable microcapsules, emulsions, sprays, aerosols, powders, biodegradable polymers, mineral oil, triglyceride oil, silicone oil, glycerol monostearate, alcohols, emulsifiers, liquid petroleum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene, wax, sorbitan monostearate, polysorbate, cetyl esters wax, cetostearyl alcohol, 2-octyldodecanol, benzyl alcohol, cyclomethicone, cyclopentasiloxane, and water. The preservative includes a preservative selected from the group consisting of tocopherol, diiodomethyl-p-tolylsulfone, 2-bromo-2-nitropropane-1, 3-diol, cis-isomer 1- (3-chloroallyl) -3, 5, 7-triaza-1-azonia adamantane chloride, glutaraldehyde, 4-dimethyloxazolidine, 7-ethylbicyclooxazolidine, methylparaben, sorbic acid, germanben ii, rosemary extract and EDTA.

The recombinant collagen of the present invention may be synthetic or may be expressed recombinantly. In the case of recombinant expression, the recombinant collagen of the present invention may be encoded by a polynucleotide. The polynucleotide may be codon optimized for the host cell in which the expression is to be performed.

In the present invention, the term "vector" is a nucleic acid vehicle into which a polynucleotide can be inserted. When a vector enables expression of a protein encoded by an inserted nucleic acid molecule, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction or transfection such that the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes, such as Yeast Artificial Chromosome (YAC), bacterial Artificial Chromosome (BAC), or P1-derived artificial chromosome (PAC); phages such as lambda phage or M13 phage, animal viruses, etc. The vector may contain a variety of elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain a replication origin. The vector may comprise a nucleic acid of the invention for ease of introduction into a cell for expression. The vector may comprise an expression control element, such as a promoter, terminator and/or enhancer, operably linked to the nucleic acid.

In the present invention, the term "host cell" is a cell into which a nucleic acid molecule has been introduced by molecular biological techniques. These techniques include transfection of viral vectors, transformation with plasmid vectors, and accelerated introduction of naked DNA by electroporation, lipofection, and particle gun.

In the present invention, the degree of relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity". For the purposes of the present invention, a software application such as the software application described in the embos package (embos: the sequence identity between two amino acid sequences was determined by the Nidel-crafts algorithm (Needleman and Wunsch,1970, J.mol. Biol. [ J.Mole. ] 48:443-453) performed by the Nidel program of European molecular biology open software suite, rice et al, 2000,Trends Genet [ genetics trend ]16:276-277, preferably version 5.0.0 or newer). The parameters used are gap opening penalty of 10, gap extension penalty of 0.5, and EBLOSUM62 (the emoss version of BLOSUM 62) substitution matrix. The output of the nitel labeled "longest identity" (obtained using the non-simplified option) was used as the percent identity and calculated as follows: (identical residues×100)/(alignment length-total number of gaps in the alignment).

For the purposes of the present invention, sequence identity between two nucleotide sequences is determined using the Nidelman-Wen application algorithm (Needleman and Wunsch,1970, supra) as implemented in the Nidel program of the EMBOSS software package (EMBOSS: european molecular biology open software suite, rice et al, 2000, supra), preferably version 5.0.0 or newer. The parameters used are gap opening penalty 10, gap extension penalty 0.5, and EDNAFULL (EMBOSS version of NCBI NUC 4.4) substitution matrix. The output of the nitel labeled "longest identity" (obtained using the non-simplified option) was used as the percent identity and calculated as follows: (identical deoxyribonucleotides. Times.100)/(alignment length-total number of gaps in the alignment).

In the present invention, the recombinant collagen of the present invention or the repeating units constituting the recombinant collagen may have a certain mutation. For example, the amino acid sequence of one or more of these moieties may have substitutions, deletions, additions or insertions of amino acid residues, i.e., the invention may use repeat unit variants, provided the variants retain collagen activity.

Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention.

Before the embodiments of the invention are explained in further detail, it is to be understood that the invention is not limited in its scope to the particular embodiments described below; it is also to be understood that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention; in the description and claims of the invention, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

Where numerical ranges are provided in the examples, it is understood that unless otherwise stated herein, both endpoints of each numerical range and any number between the two endpoints are significant both in the numerical range. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, materials used in the embodiments, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present invention may be used to practice the present invention according to the knowledge of one skilled in the art and the description of the present invention.

The sequence information used in the present invention is as follows:

SEQ ID No.1

GARGPPGPAGANGAPGLRGGAGEPGKNGAKGEPGPRGERGEAGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPP*

SEQ ID No.2

GGTGCTCGTGGACCTCCAGGTCCAGCTGGTGCAAATGGCGCACCAGGTTTAAGAGGTGGTGCTGGTGAGCCTGGTAAGAACGGTGCTAAGGGTGAGCCAGGTCCACGTGGTGAAAGAGGCGAAGCTGGTGCTAAAGGTGAAGACGGTAAAGACGGATCTCCAGGTGAACCTGGAGCTAACGGTTTACCAGGTGCAGCTGGTGAGAGAGGCGCACCTGGTTTCAGAGGTCCAGCCGGTCCAAATGGCATTCCAGGTGAGAAAGGTCCAGCAGGCGAAAGAGGTGCACCTGGACCTGCCGGACCAAGAGGTGCTGCTGGTGAACCAGGTAGAGATGGTGTACCTGGCGGACCAGGTATGAGAGGTATGCCAGGTAGTCCAGGCGGACCTGGTAGTGATGGAAAGCCTGGACCTCCAGGCAGTCAAGGTGAGTCTGGTAGGCCTGGTCCACCAGGTCCATCTGGTCCAAGAGGTCAACCAGGTCCAAAGGGTAATGACGGAGCTCCAGGCAAGAACGGAGAGAGAGGTGGTCCAGGCGGTCCAGGTCCACAGGGACCACCAGGTAAGAATGGCGAAACTGGACCACAAGGTCCACCTGGTCCAACTGGTCCAGGTGGAGATAAGGGTGATACCGGACCACCTGGTCCACAAGGTTTGCAAGGTCTACCAGGTACAGGTGGACCACCAGGCGAGAATGGTAAGCCTGGAGAACCTGGACCAAAGGGTGACGCTGGTGCTCCAGGCGCTCCAGGTGGTAAAGGTGATGCTGGAGCACCTGGCGAGAGAGGTCCACCATAG

SEQ ID No.3

GLRGGAGPPGPEGGKGAAGPPGPPGAAGERGGLGSPGPKGDKGEPGGPGADGVPGKDGPRGPTGPIGPPGPAGQPGDKGEGGAPGPRGSPGERGETGPPGPAGFPGAPGQNGEPGGKGERGAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGERGSPGGPGARGLPGPPGSNGNPGPPGPSGSPGKDGPPGPAGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGMPGPRGSPGPQGVKGESGKPGANGLSGERGPPGPQ *

SEQ ID No.4

GGTTTGAGAGGTGGAGCTGGACCACCTGGTCCAGAAGGTGGAAAGGGTGCAGCTGGTCCACCTGGACCTCCAGGTGCTGCTGGTGAACGTGGTGGTTTAGGTTCCCCTGGACCTAAAGGTGACAAAGGTGAACCAGGCGGACCAGGCGCTGATGGTGTGCCTGGTAAAGACGGTCCACGTGGTCCAACTGGTCCAATCGGTCCACCTGGACCAGCAGGTCAACCAGGTGATAAGGGTGAAGGTGGTGCTCCAGGTCCAAGAGGTAGTCCAGGCGAAAGAGGTGAAACCGGTCCACCAGGTCCAGCTGGTTTCCCTGGTGCACCTGGTCAGAATGGTGAACCTGGTGGTAAGGGAGAAAGAGGCGCACCAGGTGAGAAAGGTGAAGGCGGACCTCCAGGCGTTGCCGGACCACCAGGCGGTTCTGGACCTGCTGGACCACCTGGACCACAAGGTGTAAAGGGTGAGAGAGGTTCACCTGGCGGTCCAGGCGCAAGAGGTTTGCCTGGTCCACCAGGCTCTAACGGCAATCCAGGTCCACCAGGTCCATCTGGTTCTCCAGGTAAAGATGGTCCACCTGGTCCAGCCGGTAATACCGGAGCACCTGGATCACCTGGAGTTTCTGGTCCAAAGGGTGATGCTGGTCAACCTGGTGAGAAGGGATCTCCAGGCGCTCAAGGTCCACCTGGCGCTCCAGGTATGCCTGGACCAAGAGGCTCACCTGGACCTCAAGGTGTTAAAGGCGAATCTGGTAAACCAGGTGCTAATGGCTTGTCTGGAGAGAGAGGTCCACCAGGTCCACAATAG

SEQ ID No.5

GARGNDGARGSDGQPGPPGPPGTAGFPGSPGAKGEVGPAGSPGSNGAPGQRGEPGPQGHAGAQGPPGPPGINGSPGGKGEMGARGPPGPAGANGAPGLRGGAGEPGKNGAKGEPGPRGERGEAGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQP*

SEQ ID No.6

GGTGCTAGAGGTAATGACGGAGCTAGAGGTTCTGATGGTCAACCTGGTCCACCAGGTCCACCTGGTACTGCTGGATTTCCAGGTTCACCTGGTGCAAAGGGTGAAGTTGGACCTGCAGGTTCTCCAGGCTCTAATGGCGCTCCAGGTCAGAGAGGTGAGCCAGGTCCACAAGGTCATGCTGGTGCTCAAGGTCCACCTGGACCACCTGGCATCAACGGATCACCAGGTGGTAAGGGTGAAATGGGTGCAAGAGGTCCACCTGGTCCAGCAGGTGCTAATGGAGCACCAGGTTTGAGAGGTGGTGCTGGAGAGCCAGGTAAGAACGGTGCTAAGGGTGAGCCTGGTCCACGTGGTGAAAGAGGTGAAGCTGGTGCTAAAGGTGAAGACGGTAAAGACGGATCTCCAGGCGAACCTGGAGCAAACGGTTTACCAGGTGCAGCTGGAGAACGAGGTGCTCCAGGCTTTCGTGGACCTGCTGGTCCAAACGGTATTCCAGGTGAGAAAGGTCCAGCTGGTGAGAGAGGTGCACCTGGACCTGCCGGACCTAGAGGTGCTGCTGGTGAACCAGGTAGAGATGGTGTTCCAGGCGGACCAGGTATGAGAGGTATGCCTGGTAGTCCAGGCGGTCCAGGTAGTGATGGTAAGCCTGGACCACCAGGTAGTCAAGGTGAGTCTGGTAGGCCTGGACCTCCAGGTCCATCTGGTCCAAGAGGTCAACCATAG

SEQ ID NO.7

AGATCTAACATCCAAAGACGAAAGGTTGAATGAAACCTTTTTGCCATCCGACATCCACAGGTCCATTCTCACACATAAGTGCCAAACGCAACAGGAGGGGATACACTAGCAGCAGACCGTTGCAAACGCAGGACCTCCACTCCTCTTCTCCTCAACACCCACTTTTGCCATCGAAAAACCAGCCCAGTTATTGGGCTTGATTGGAGCTCGCTCATTCCAATTCCTTCTATTAGGCTACTAACACCATGACTTTATTAGCCTGTCTATCCTGGCCCCCCTGGCGAGGTTCATGTTTGTTTATTTCCGAATGCAACAAGCTCCGCATTACACCCGAACATCACTCCAGATGAGGGCTTTCTGAGTGTGGGGTCAAATAGTTTCATGTTCCCCAAATGGCCCAAAACTGACAGTTTAAACGCTGTCTTGGAACCTAATATGACAAAAGCGTGATCTCATCCAAGATGAACTAAGTTTGGTTCGTTGAAATGCTAACGGCCAGTTGGTCAAAAAGAAACTTCCAAAAGTCGCCATACCGTTTGTCTTGTTTGGTATTGATTGACGAATGCTCAAAAATAATCTCATTAATGCTTAGCGCAGTCTCTCTATCGCTTCTGAACCCCGGTGCACCTGTGCCGAAACGCAAATGGGGAAACACCCGCTTTTTGGATGATTATGCATTGTCTCCACATTGTATGCTTCCAAGATTCTGGTGGGAATACTGCTGATAGCCTAACGTTCATGATCAAAATTTAACTGTTCTAACCCCTACTTGACAGCAATATATAAACAGAAGGAAGCTGCCCTGTCTTAAACCTTTTTTTTTATCATCATTATTAGCTTACTTTCATAATTGCGACTGGTTCCAATTGACAAGCTTTTGATTTTAACGACTTTTAACGACAACTTGAGAAGATCAAAAAACAACTAATTATTCGAAGGATCCAAACGATGAGATTTCCTTCAATTTTTACTGCAGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTCCAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTACTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTCTCGAGAAAAGAGAGGCTGAAGCTTACGTAGAATTCCCTAGGGCGGCCGCGAATTAATTCGCCTTAGACATGACTGTTCCTCAGTTCAAGTTGGGCACTTACGAGAAGACCGGTCTTGCTAGATTCTAATCAAGAGGATGTCAGAATGCCATTTGCCTGAGAGATGCAGGCTTCATTTTTGATACTTTTTTATTTGTAACCTATATAGTATAGGATTTTTTTTGTCATTTTGTTTCTTCTCGTACGAGCTTGCTCCTGATCAGCCTATCTCGCAGCTGATGAATATCTTGTGGTAGGGGTTTGGGAAAATCATTCGAGTTTGATGTTTTTCTTGGTATTTCCCACTCCTCTTCAGAGTACAGAAGATTAAGTGAGAAGTTCGTTTGTGCAAGCTTATCGATAAGCTTTAATGCGGTAGTTTATCACAGTTAAATTGCTAACGCAGTCAGGCACCGTGTATGAAATCTAACAATGCGCTCATCGTCATCCTCGGCACCGTCACCCTGGATGCTGTAGGCATAGGCTTGGTTATGCCGGTACTGCCGGGCCTCTTGCGGGATATCGTCCATTCCGACAGCATCGCCAGTCACTATGGCGTGCTGCTAGCGCTATATGCGTTGATGCAATTTCTATGCGCACCCGTTCTCGGAGCACTGTCCGACCGCTTTGGCCGCCGCCCAGTCCTGCTCGCTTCGCTACTTGGAGCCACTATCGACTACGCGATCATGGCGACCACACCCGTCCTGTGGATCTATCGAATCTAAATGTAAGTTAAAATCTCTAAATAATTAAATAAGTCCCAGTTTCTCCATACGAACCTTAACAGCATTGCGGTGAGCATCTAGACCTTCAACAGCAGCCAGATCCATCACTGCTTGGCCAATATGTTTCAGTCCCTCAGGAGTTACGTCTTGTGAAGTGATGAACTTCTGGAAGGTTGCAGTGTTAACTCCGCTGTATTGACGGGCATATCCGTACGTTGGCAAAGTGTGGTTGGTACCGGAGGAGTAATCTCCACAACTCTCTGGAGAGTAGGCACCAACAAACACAGATCCAGCGTGTTGTACTTGATCAACATAAGAAGAAGCATTCTCGATTTGCAGGATCAAGTGTTCAGGAGCGTACTGATTGGACATTTCCAAAGCCTGCTCGTAGGTTGCAACCGATAGGGTTGTAGAGTGTGCAATACACTTGCGTACAATTTCAACCCTTGGCAACTGCACAGCTTGGTTGTGAACAGCATCTTCAATTCTGGCAAGCTCCTTGTCTGTCATATCGACAGCCAACAGAATCACCTGGGAATCAATACCATGTTCAGCTTGAGACAGAAGGTCTGAGGCAACGAAATCTGGATCAGCGTATTTATCAGCAATAACTAGAACTTCAGAAGGCCCAGCAGGCATGTCAATACTACACAGGGCTGATGTGTCATTTTGAACCATCATCTTGGCAGCAGTAACGAACTGGTTTCCTGGACCAAATATTTTGTCACACTTAGGAACAGTTTCTGTTCCGTAAGCCATAGCAGCTACTGCCTGGGCGCCTCCTGCTAGCACGATACACTTAGCACCAACCTTGTGGGCAACGTAGATGACTTCTGGGGTAAGGGTACCATCCTTCTTAGGTGGAGATGCAAAAACAATTTCTTTGCAACCAGCAACTTTGGCAGGAACACCCAGCATCAGGGAAGTGGAAGGCAGAATTGCGGTTCCACCAGGAATATAGAGGCCAACTTTCTCAATAGGTCTTGCAAAACGAGAGCAGACTACACCAGGGCAAGTCTCAACTTGCAACGTCTCCGTTAGTTGAGCTTCATGGAATTTCCTGACGTTATCTATAGAGAGATCAATGGCTCTCTTAACGTTATCTGGCAATTGCATAAGTTCCTCTGGGAAAGGAGCTTCTAACACAGGTGTCTTCAAAGCGACTCCATCAAACTTGGCAGTTAGTTCTAAAAGGGCTTTGTCACCATTTTGACGAACATTGTCGACAATTGGTTTGACTAATTCCATAATCTGTTCCGTTTTCTGGATAGGACGACGAAGGGCATCTTCAATTTCTTGTGAGGAGGCCTTAGAAACGTCAATTTTGCACAATTCAATACGACCTTCAGAAGGGACTTCTTTAGGTTTGGATTCTTCTTTAGGTTGTTCCTTGGTGTATCCTGGCTTGGCATCTCCTTTCCTTCTAGTGACCTTTAGGGACTTCATATCCAGGTTTCTCTCCACCTCGTCCAACGTCACACCGTACTTGGCACATCTAACTAATGCAAAATAAAATAAGTCAGCACATTCCCAGGCTATATCTTCCTTGGATTTAGCTTCTGCAAGTTCATCAGCTTCCTCCCTAATTTTAGCGTTCAACAAAACTTCGTCGTCAAATAACCGTTTGGTATAAGAACCTTCTGGAGCATTGCTCTTACGATCCCACAAGGTGGCTTCCATGGCTCTAAGACCCTTTGATTGGCCAAAACAGGAAGTGCGTTCCAAGTGACAGAAACCAACACCTGTTTGTTCAACCACAAATTTCAAGCAGTCTCCATCACAATCCAATTCGATACCCAGCAACTTTTGAGTTGCTCCAGATGTAGCACCTTTATACCACAAACCGTGACGACGAGATTGGTAGACTCCAGTTTGTGTCCTTATAGCCTCCGGAATAGACTTTTTGGACGAGTACACCAGGCCCAACGAGTAATTAGAAGAGTCAGCCACCAAAGTAGTGAATAGACCATCGGGGCGGTCAGTAGTCAAAGACGCCAACAAAATTTCACTGACAGGGAACTTTTTGACATCTTCAGAAAGTTCGTATTCAGTAGTCAATTGCCGAGCATCAATAATGGGGATTATACCAGAAGCAACAGTGGAAGTCACATCTACCAACTTTGCGGTCTCAGAAAAAGCATAAACAGTTCTACTACCGCCATTAGTGAAACTTTTCAAATCGCCCAGTGGAGAAGAAAAAGGCACAGCGATACTAGCATTAGCGGGCAAGGATGCAACTTTATCAACCAGGGTCCTATAGATAACCCTAGCGCCTGGGATCATCCTTTGGACAACTCTTTCTGCCAAATCTAGGTCCAAAATCACTTCATTGATACCATTATTGTACAACTTGAGCAAGTTGTCGATCAGCTCCTCAAATTGGTCCTCTGTAACGGATGACTCAACTTGCACATTAACTTGAAGCTCAGTCGATTGAGTGAACTTGATCAGGTTGTGCAGCTGGTCAGCAGCATAGGGAAACACGGCTTTTCCTACCAAACTCAAGGAATTATCAAACTCTGCAACACTTGCGTATGCAGGTAGCAAGGGAAATGTCATACTTGAAGTCGGACAGTGAGTGTAGTCTTGAGAAATTCTGAAGCCGTATTTTTATTATCAGTGAGTCAGTCATCAGGAGATCCTCTACGCCGGACGCATCGTGGCCGACCTGCAGGGGGGGGGGGGGCGCTGAGGTCTGCCTCGTGAAGAAGGTGTTGCTGACTCATACCAGGCCTGAATCGCCCCATCATCCAGCCAGAAAGTGAGGGAGCCACGGTTGATGAGAGCTTTGTTGTAGGTGGACCAGTTGGTGATTTTGAACTTTTGCTTTGCCACGGAACGGTCTGCGTTGTCGGGAAGATGCGTGATCTGATCCTTCAACTCAGCAAAAGTTCGATTTATTCAACAAAGCCGCCGTCCCGTCAAGTCAGCGTAATGCTCTGCCAGTGTTACAACCAATTAACCAATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTCGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGGCTTTCCCCCCCCCCCCTGCAGGTCGGCATCACCGGCGCCACAGGTGCGGTTGCTGGCGCCTATATCGCCGACATCACCGATGGGGAAGATCGGGCTCGCCACTTCGGGCTCATGAGCGCTTGTTTCGGCGTGGGTATGGTGGCAGGCCCCGTGGCCGGGGGACTGTTGGGCGCCATCTCCTTGCATGCACCATTCCTTGCGGCGGCGGTGCTCAACGGCCTCAACCTACTACTGGGCTGCTTCCTAATGCAGGAGTCGCATAAGGGAGAGCGTCGAGTATCTATGATTGGAAGTATGGGAATGGTGATACCCGCATTCTTCAGTGTCTTGAGGTCTCCTATCAGATTATGCCCAACTAAAGCAACCGGAGGAGGAGATTTCATGGTAAATTTCTCTGACTTTTGGTCATCAGTAGACTCGAACTGTGAGACTATCTCGGTTATGACAGCAGAAATGTCCTTCTTGGAGACAGTAAATGAAGTCCCACCAATAAAGAAATCCTTGTTATCAGGAACAAACTTCTTGTTTCGAACTTTTTCGGTGCCTTGAACTATAAAATGTAGAGTGGATATGTCGGGTAGGAATGGAGCGGGCAAATGCTTACCTTCTGGACCTTCAAGAGGTATGTAGGGTTTGTAGATACTGATGCCAACTTCAGTGACAACGTTGCTATTTCGTTCAAACCATTCCGAATCCAGAGAAATCAAAGTTGTTTGTCTACTATTGATCCAAGCCAGTGCGGTCTTGAAACTGACAATAGTGTGCTCGTGTTTTGAGGTCATCTTTGTATGAATAAATCTAGTCTTTGATCTAAATAATCTTGACGAGCCAAGGCGATAAATACCCAAATCTAAAACTCTTTTAAAACGTTAAAAGGACAAGTATGTCTGCCTGTATTAAACCCCAAATCAGCTCGTAGTCTGATCCTCATCAACTTGAGGGGCACTATCTTGTTTTAGAGAAATTTGCGGAGATGCGATATCGAGAAAAAGGTACGCTGATTTTAAACGTGAAATTTATCTCAAGATCTCTGCCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGACCCAGTCACGTAGCGATAGCGGAGTGTATACTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTGCAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAACACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTTCAAGAATTAATTCTCATGTTTGACAGCTTATCATCGATAAGCTGACTCATGTTGGTATTGTGAAATAGACGCAGATCGGGAACACTGAAAAATAACAGTTATTATTCG

SEQ ID No.8

GLRGGAGPPGPEGGKGAAGPPGPPGAAGERGGLGSPGPKGDKGEPGGPGADGVPGKDGPRGPTGPIGPPGPAGQPGDKGEGGAPGPRGSPGERGETGPPGPAGFPGAPGQNGEPGGKGERGAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGERGSPGGPGARGLPGPPGSNGNPGPPGPSGSPGKDGPPGPAGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGMPGPRGSPGPQGVKGESGKPGANGLSGERGPPGPQGARGPPGPAGANGAPGLRGGAGEPGKNGAKGEPGPRGERGEAGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPGARGNDGARGSDGQPGPPGPPGTAGFPGSPGAKGEVGPAGSPGSNGAPGQRGEPGPQGHAGAQGPPGPPGINGSPGGKGEMGARGPPGPAGANGAPGLRGGAGEPGKNGAKGEPGPRGERGEAGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQP*

SEQ ID No.9

GGTTTGAGAGGTGGAGCTGGACCACCTGGTCCAGAAGGTGGAAAGGGTGCAGCTGGTCCACCTGGACCTCCAGGTGCTGCTGGTGAACGTGGTGGTTTAGGTTCCCCTGGACCTAAAGGTGACAAAGGTGAACCAGGCGGACCAGGCGCTGATGGTGTGCCTGGTAAAGACGGTCCACGTGGTCCAACTGGTCCAATCGGTCCACCTGGACCAGCAGGTCAACCAGGTGATAAGGGTGAAGGTGGTGCTCCAGGTCCAAGAGGTAGTCCAGGCGAAAGAGGTGAAACCGGTCCACCAGGTCCAGCTGGTTTCCCTGGTGCACCTGGTCAGAATGGTGAACCTGGTGGTAAGGGAGAAAGAGGCGCACCAGGTGAGAAAGGTGAAGGCGGACCTCCAGGCGTTGCCGGACCACCAGGCGGTTCTGGACCTGCTGGACCACCTGGACCACAAGGTGTAAAGGGTGAGAGAGGTTCACCTGGCGGTCCAGGCGCAAGAGGTTTGCCTGGTCCACCAGGCTCTAACGGCAATCCAGGTCCACCAGGTCCATCTGGTTCTCCAGGTAAAGATGGTCCACCTGGTCCAGCCGGTAATACCGGAGCACCTGGATCACCTGGAGTTTCTGGTCCAAAGGGTGATGCTGGTCAACCTGGTGAGAAGGGATCTCCAGGCGCTCAAGGTCCACCTGGCGCTCCAGGTATGCCTGGACCAAGAGGCTCACCTGGACCTCAAGGTGTTAAAGGCGAATCTGGTAAACCAGGTGCTAATGGCTTGTCTGGAGAGAGAGGTCCACCAGGTCCACAAGGTGCTCGTGGACCTCCAGGTCCAGCTGGTGCAAATGGCGCACCAGGTTTAAGAGGTGGTGCTGGTGAGCCTGGTAAGAACGGTGCTAAGGGTGAGCCAGGTCCACGTGGTGAAAGAGGCGAAGCTGGTGCTAAAGGTGAAGACGGTAAAGACGGATCTCCAGGTGAACCTGGAGCTAACGGTTTACCAGGTGCAGCTGGTGAGAGAGGCGCACCTGGTTTCAGAGGTCCAGCCGGTCCAAATGGCATTCCAGGTGAGAAAGGTCCAGCAGGCGAAAGAGGTGCACCTGGACCTGCCGGACCAAGAGGTGCTGCTGGTGAACCAGGTAGAGATGGTGTACCTGGCGGACCAGGTATGAGAGGTATGCCAGGTAGTCCAGGCGGACCTGGTAGTGATGGAAAGCCTGGACCTCCAGGCAGTCAAGGTGAGTCTGGTAGGCCTGGTCCACCAGGTCCATCTGGTCCAAGAGGTCAACCAGGTCCAAAGGGTAATGACGGAGCTCCAGGCAAGAACGGAGAGAGAGGTGGTCCAGGCGGTCCAGGTCCACAGGGACCACCAGGTAAGAATGGCGAAACTGGACCACAAGGTCCACCTGGTCCAACTGGTCCAGGTGGAGATAAGGGTGATACCGGACCACCTGGTCCACAAGGTTTGCAAGGTCTACCAGGTACAGGTGGACCACCAGGCGAGAATGGTAAGCCTGGAGAACCTGGACCAAAGGGTGACGCTGGTGCTCCAGGCGCTCCAGGTGGTAAAGGTGATGCTGGAGCACCTGGCGAGAGAGGTCCACCAGGTGCTAGAGGTAATGACGGAGCTAGAGGTTCTGATGGTCAACCTGGTCCACCAGGTCCACCTGGTACTGCTGGATTTCCAGGTTCACCTGGTGCAAAGGGTGAAGTTGGACCTGCAGGTTCTCCAGGCTCTAATGGCGCTCCAGGTCAGAGAGGTGAGCCAGGTCCACAAGGTCATGCTGGTGCTCAAGGTCCACCTGGACCACCTGGCATCAACGGATCACCAGGTGGTAAGGGTGAAATGGGTGCAAGAGGTCCACCTGGTCCAGCAGGTGCTAATGGAGCACCAGGTTTGAGAGGTGGTGCTGGAGAGCCAGGTAAGAACGGTGCTAAGGGTGAGCCTGGTCCACGTGGTGAAAGAGGTGAAGCTGGTGCTAAAGGTGAAGACGGTAAAGACGGATCTCCAGGCGAACCTGGAGCAAACGGTTTACCAGGTGCAGCTGGAGAACGAGGTGCTCCAGGCTTTCGTGGACCTGCTGGTCCAAACGGTATTCCAGGTGAGAAAGGTCCAGCTGGTGAGAGAGGTGCACCTGGACCTGCCGGACCTAGAGGTGCTGCTGGTGAACCAGGTAGAGATGGTGTTCCAGGCGGACCAGGTATGAGAGGTATGCCTGGTAGTCCAGGCGGTCCAGGTAGTGATGGTAAGCCTGGACCACCAGGTAGTCAAGGTGAGTCTGGTAGGCCTGGACCTCCAGGTCCATCTGGTCCAAGAGGTCAACCATAG

SEQ ID No.10

GLRGGAGPPGPEGGKGAAGPPGPPGAAGERGGLGSPGPKGDKGEPGGPGADGVPGKDGPRGPTGPIGPPGPAGQPGDKGEGGAPGPRGSPGERGETGPPGPAGFPGAPGQNGEPGGKGERGAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGERGSPGGPGARGLPGPPGSNGNPGPPGPSGSPGKDGPPGPAGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGMPGPRGSPGPQGVKGESGKPGANGLSGERGPPGPQGARGPPGPAGANGAPGLRGGAGEPGKNGAKGEPGPRGERGEAGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPGARGNDGARGSDGQPGPPGPPGTAGFPGSPGAKGEVGPAGSPGSNGAPGQRGEPGPQGHAGAQGPPGPPGINGSPGGKGEMGARGPPGPAGANGAPGLRGGAGEPGKNGAKGEPGPRGERGEAGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQPGLRGGAGPPGPEGGKGAAGPPGPPGAAGERGGLGSPGPKGDKGEPGGPGADGVPGKDGPRGPTGPIGPPGPAGQPGDKGEGGAPGPRGSPGERGETGPPGPAGFPGAPGQNGEPGGKGERGAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGERGSPGGPGARGLPGPPGSNGNPGPPGPSGSPGKDGPPGPAGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGMPGPRGSPGPQGVKGESGKPGANGLSGERGPPGPQGARGPPGPAGANGAPGLRGGAGEPGKNGAKGEPGPRGERGEAGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPGARGNDGARGSDGQPGPPGPPGTAGFPGSPGAKGEVGPAGSPGSNGAPGQRGEPGPQGHAGAQGPPGPPGINGSPGGKGEMGARGPPGPAGANGAPGLRGGAGEPGKNGAKGEPGPRGERGEAGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQP*

SEQ ID No.11

GGTTTGAGAGGTGGAGCTGGACCACCTGGTCCAGAAGGTGGAAAGGGTGCAGCTGGTCCACCTGGACCTCCAGGTGCTGCTGGTGAACGTGGTGGTTTAGGTTCCCCTGGACCTAAAGGTGACAAAGGTGAACCAGGCGGACCAGGCGCTGATGGTGTGCCTGGTAAAGACGGTCCACGTGGTCCAACTGGTCCAATCGGTCCACCTGGACCAGCAGGTCAACCAGGTGATAAGGGTGAAGGTGGTGCTCCAGGTCCAAGAGGTAGTCCAGGCGAAAGAGGTGAAACCGGTCCACCAGGTCCAGCTGGTTTCCCTGGTGCACCTGGTCAGAATGGTGAACCTGGTGGTAAGGGAGAAAGAGGCGCACCAGGTGAGAAAGGTGAAGGCGGACCTCCAGGCGTTGCCGGACCACCAGGCGGTTCTGGACCTGCTGGACCACCTGGACCACAAGGTGTAAAGGGTGAGAGAGGTTCACCTGGCGGTCCAGGCGCAAGAGGTTTGCCTGGTCCACCAGGCTCTAACGGCAATCCAGGTCCACCAGGTCCATCTGGTTCTCCAGGTAAAGATGGTCCACCTGGTCCAGCCGGTAATACCGGAGCACCTGGATCACCTGGAGTTTCTGGTCCAAAGGGTGATGCTGGTCAACCTGGTGAGAAGGGATCTCCAGGCGCTCAAGGTCCACCTGGCGCTCCAGGTATGCCTGGACCAAGAGGCTCACCTGGACCTCAAGGTGTTAAAGGCGAATCTGGTAAACCAGGTGCTAATGGCTTGTCTGGAGAGAGAGGTCCACCAGGTCCACAAGGTGCTCGTGGACCTCCAGGTCCAGCTGGTGCAAATGGCGCACCAGGTTTAAGAGGTGGTGCTGGTGAGCCTGGTAAGAACGGTGCTAAGGGTGAGCCAGGTCCACGTGGTGAAAGAGGCGAAGCTGGTGCTAAAGGTGAAGACGGTAAAGACGGATCTCCAGGTGAACCTGGAGCTAACGGTTTACCAGGTGCAGCTGGTGAGAGAGGCGCACCTGGTTTCAGAGGTCCAGCCGGTCCAAATGGCATTCCAGGTGAGAAAGGTCCAGCAGGCGAAAGAGGTGCACCTGGACCTGCCGGACCAAGAGGTGCTGCTGGTGAACCAGGTAGAGATGGTGTACCTGGCGGACCAGGTATGAGAGGTATGCCAGGTAGTCCAGGCGGACCTGGTAGTGATGGAAAGCCTGGACCTCCAGGCAGTCAAGGTGAGTCTGGTAGGCCTGGTCCACCAGGTCCATCTGGTCCAAGAGGTCAACCAGGTCCAAAGGGTAATGACGGAGCTCCAGGCAAGAACGGAGAGAGAGGTGGTCCAGGCGGTCCAGGTCCACAGGGACCACCAGGTAAGAATGGCGAAACTGGACCACAAGGTCCACCTGGTCCAACTGGTCCAGGTGGAGATAAGGGTGATACCGGACCACCTGGTCCACAAGGTTTGCAAGGTCTACCAGGTACAGGTGGACCACCAGGCGAGAATGGTAAGCCTGGAGAACCTGGACCAAAGGGTGACGCTGGTGCTCCAGGCGCTCCAGGTGGTAAAGGTGATGCTGGAGCACCTGGCGAGAGAGGTCCACCAGGTGCTAGAGGTAATGACGGAGCTAGAGGTTCTGATGGTCAACCTGGTCCACCAGGTCCACCTGGTACTGCTGGATTTCCAGGTTCACCTGGTGCAAAGGGTGAAGTTGGACCTGCAGGTTCTCCAGGCTCTAATGGCGCTCCAGGTCAGAGAGGTGAGCCAGGTCCACAAGGTCATGCTGGTGCTCAAGGTCCACCTGGACCACCTGGCATCAACGGATCACCAGGTGGTAAGGGTGAAATGGGTGCAAGAGGTCCACCTGGTCCAGCAGGTGCTAATGGAGCACCAGGTTTGAGAGGTGGTGCTGGAGAGCCAGGTAAGAACGGTGCTAAGGGTGAGCCTGGTCCACGTGGTGAAAGAGGTGAAGCTGGTGCTAAAGGTGAAGACGGTAAAGACGGATCTCCAGGCGAACCTGGAGCAAACGGTTTACCAGGTGCAGCTGGAGAACGAGGTGCTCCAGGCTTTCGTGGACCTGCTGGTCCAAACGGTATTCCAGGTGAGAAAGGTCCAGCTGGTGAGAGAGGTGCACCTGGACCTGCCGGACCTAGAGGTGCTGCTGGTGAACCAGGTAGAGATGGTGTTCCAGGCGGACCAGGTATGAGAGGTATGCCTGGTAGTCCAGGCGGTCCAGGTAGTGATGGTAAGCCTGGACCACCAGGTAGTCAAGGTGAGTCTGGTAGGCCTGGACCTCCAGGTCCATCTGGTCCAAGAGGTCAACCAGGTTTGAGAGGTGGAGCTGGACCACCTGGTCCAGAAGGTGGAAAGGGTGCAGCTGGTCCACCTGGACCTCCAGGTGCTGCTGGTGAACGTGGTGGTTTAGGTTCCCCTGGACCTAAAGGTGACAAAGGTGAACCAGGCGGACCAGGCGCTGATGGTGTGCCTGGTAAAGACGGTCCACGTGGTCCAACTGGTCCAATCGGTCCACCTGGACCAGCAGGTCAACCAGGTGATAAGGGTGAAGGTGGTGCTCCAGGTCCAAGAGGTAGTCCAGGCGAAAGAGGTGAAACCGGTCCACCAGGTCCAGCTGGTTTCCCTGGTGCACCTGGTCAGAATGGTGAACCTGGTGGTAAGGGAGAAAGAGGCGCACCAGGTGAGAAAGGTGAAGGCGGACCTCCAGGCGTTGCCGGACCACCAGGCGGTTCTGGACCTGCTGGACCACCTGGACCACAAGGTGTAAAGGGTGAGAGAGGTTCACCTGGCGGTCCAGGCGCAAGAGGTTTGCCTGGTCCACCAGGCTCTAACGGCAATCCAGGTCCACCAGGTCCATCTGGTTCTCCAGGTAAAGATGGTCCACCTGGTCCAGCCGGTAATACCGGAGCACCTGGATCACCTGGAGTTTCTGGTCCAAAGGGTGATGCTGGTCAACCTGGTGAGAAGGGATCTCCAGGCGCTCAAGGTCCACCTGGCGCTCCAGGTATGCCTGGACCAAGAGGCTCACCTGGACCTCAAGGTGTTAAAGGCGAATCTGGTAAACCAGGTGCTAATGGCTTGTCTGGAGAGAGAGGTCCACCAGGTCCACAAGGTGCTCGTGGACCTCCAGGTCCAGCTGGTGCAAATGGCGCACCAGGTTTAAGAGGTGGTGCTGGTGAGCCTGGTAAGAACGGTGCTAAGGGTGAGCCAGGTCCACGTGGTGAAAGAGGCGAAGCTGGTGCTAAAGGTGAAGACGGTAAAGACGGATCTCCAGGTGAACCTGGAGCTAACGGTTTACCAGGTGCAGCTGGTGAGAGAGGCGCACCTGGTTTCAGAGGTCCAGCCGGTCCAAATGGCATTCCAGGTGAGAAAGGTCCAGCAGGCGAAAGAGGTGCACCTGGACCTGCCGGACCAAGAGGTGCTGCTGGTGAACCAGGTAGAGATGGTGTACCTGGCGGACCAGGTATGAGAGGTATGCCAGGTAGTCCAGGCGGACCTGGTAGTGATGGAAAGCCTGGACCTCCAGGCAGTCAAGGTGAGTCTGGTAGGCCTGGTCCACCAGGTCCATCTGGTCCAAGAGGTCAACCAGGTCCAAAGGGTAATGACGGAGCTCCAGGCAAGAACGGAGAGAGAGGTGGTCCAGGCGGTCCAGGTCCACAGGGACCACCAGGTAAGAATGGCGAAACTGGACCACAAGGTCCACCTGGTCCAACTGGTCCAGGTGGAGATAAGGGTGATACCGGACCACCTGGTCCACAAGGTTTGCAAGGTCTACCAGGTACAGGTGGACCACCAGGCGAGAATGGTAAGCCTGGAGAACCTGGACCAAAGGGTGACGCTGGTGCTCCAGGCGCTCCAGGTGGTAAAGGTGATGCTGGAGCACCTGGCGAGAGAGGTCCACCAGGTGCTAGAGGTAATGACGGAGCTAGAGGTTCTGATGGTCAACCTGGTCCACCAGGTCCACCTGGTACTGCTGGATTTCCAGGTTCACCTGGTGCAAAGGGTGAAGTTGGACCTGCAGGTTCTCCAGGCTCTAATGGCGCTCCAGGTCAGAGAGGTGAGCCAGGTCCACAAGGTCATGCTGGTGCTCAAGGTCCACCTGGACCACCTGGCATCAACGGATCACCAGGTGGTAAGGGTGAAATGGGTGCAAGAGGTCCACCTGGTCCAGCAGGTGCTAATGGAGCACCAGGTTTGAGAGGTGGTGCTGGAGAGCCAGGTAAGAACGGTGCTAAGGGTGAGCCTGGTCCACGTGGTGAAAGAGGTGAAGCTGGTGCTAAAGGTGAAGACGGTAAAGACGGATCTCCAGGCGAACCTGGAGCAAACGGTTTACCAGGTGCAGCTGGAGAACGAGGTGCTCCAGGCTTTCGTGGACCTGCTGGTCCAAACGGTATTCCAGGTGAGAAAGGTCCAGCTGGTGAGAGAGGTGCACCTGGACCTGCCGGACCTAGAGGTGCTGCTGGTGAACCAGGTAGAGATGGTGTTCCAGGCGGACCAGGTATGAGAGGTATGCCTGGTAGTCCAGGCGGTCCAGGTAGTGATGGTAAGCCTGGACCACCAGGTAGTCAAGGTGAGTCTGGTAGGCCTGGACCTCCAGGTCCATCTGGTCCAAGAGGTCAACCATAG

EXAMPLE 1 construction of monomeric collagen Strain and shake flask fermentation

Gene screening:

the natural human III type collagen amino acid sequence Nature (GenBank: AGL 34959.1) is screened from NCBI database for hydrophobicity analysis, and the amino acid sequence with strong water solubility and stability is selected, and the integrated novel protein is the monomer gene B/C/E of the recombinant collagen strain, and the amino acid sequence is shown as SEQ ID NO. 1/SEQ ID NO. 3/SEQ ID NO. 5.

Constructing a host bacterium for expressing collagen:

the coding gene of the integrated monomer gene B/C/E is synthesized by Jiangsu Saxifraga flyaway biotechnology Co., ltd, cloned into plasmid pPIC9K, and the nucleotide sequence shown as SEQ ID NO.7 is formed into a recombinant vector (prepared by Shanghai Yi Isatis medicine technology Co., ltd. Biological part laboratory) which is prepared bySacAnd I, converting the GS115 host after enzyme digestion linearization, and obtaining corresponding engineering bacteria after antibiotic screening. The specific operation steps are as follows:

(1) Electric transfer Pichia pastoris cell GS115

Ice bath electric cup, 10L of linearized plasmid was added to a 1.5 mL EP tube containing 80. Mu.L pichia pastoris competent cells, mixed well and transferred to an electric cup with a diameter of 0.2 cm, and then the electric cup was ice-bathed for 5 min. The electric shock conditions are as follows: voltage 1.5 kV; capacitance 25 μF; the resistance is 200 omega, and the electric shock time is 4-10 msec. After the shock was completed, 650 μl of pre-chilled 1M sorbitol solution on ice was added to the shock conversion cup and gently blown with a gun head to homogenize the solution. All liquid in the electrorotor was transferred to a new 2 mL EP tube and incubated at 30℃for 2 h. The cells were collected by low-speed centrifugation and all the cells were plated on MD plates (composition shown in FIG. 11) and incubated at 30℃for 3 to 4℃for d.

(2) Antibiotic screening

When the colony grows out of the plate, picking single bacteria growing on the plate by using an inoculating loop, and photocopying the single bacteria on a YPD solid plate (composition is shown as figure 12) containing 0.5 g/L-4 g/L G418, and culturing at a constant temperature of 30 ℃ for 3-4 d.

(3) Small test expression identification

Induction of expression: single colonies tolerant to 4 g/L G418 were inoculated into medium containing 5 mLYPD at 30℃and cultured overnight at 220 rpm, transferred to a conical flask containing 10 mL BMGY (composition shown in FIG. 9) at 0.01% of the inoculum size, cultured overnight at 30℃and cultured at 220 rpm, and shaken to OD ₆₀₀ =2 to 6 (logarithmic growth, about 16 to 18 h); centrifugation at 5000 rpm for 5 min at room temperature, collecting cells, removing supernatant, and resuspending cells to OD with BMMY (composition shown in FIG. 10) ₆₀₀ 1, performing induction expression; adding methanol into each 24-h part to a final concentration of 1%, and continuously inducing for 96-h total time, wherein collagen can be produced at high yield, and SDS-PAGE is shown in figure 1;

EXAMPLE 2 construction of the orthotropic tandem fragment and Strain

Recombinant plasmid pPIC9K-B adopts CaC1 ₂ Transforming escherichia coli DH5 alpha by a method, screening by an LB plate containing Kan to obtain recombinant escherichia coli containing plasmid pPIC9K-B, namely DH5 alpha/pPIC 9K-B, culturing DH5 alpha/pPIC 9K-B in a liquid LB culture medium overnight to extract plasmids, obtaining a large amount of pPIC9K-B, carrying out PCR nucleic acid electrophoresis by using a primer (B-F+B-R, the nucleotide sequence of which is shown as SEQ ID No.12 and 13) containing a fragment B, cutting gel, and recovering to obtain fragments with 2 repetition and 3 repetition;

SEQ ID No.12:B-F CTGGCGAGAGAGGTCCACCAGGTGCTCGTGGACCTCCAGG

SEQ ID No.13:B-R CCTGGAGGTCCACGAGCACCTGGTGGACCTCTCTCGCCAG

3 repeated fragments are subjected to PCR by using a primer (B-F9K+B-R9K, nucleotide sequences are shown as SEQ ID No.14 and 15) containing a carrier fragment, and recombined to linearized pPIC9K #EcoRI) ObtainingTo the strain pPIC9K-3*E, plasmid extraction yielded pPIC9K-3*B.

SEQ ID No.14:B-F9K GAGGCTGAAGCTTACGTAgaattcGGTGCTCGTGGACCTC

SEQ ID No.15:B-R9K CTAAGGCGAATTAATTCGCGGCCGCctaTGGTGGACCTCTC TCGCC

2 repeated fragments and 3 repeated fragments obtained by PCR are subjected to PCR by using a primer (B-F9K+B-R, the nucleotide sequences of which are shown as SEQ ID No.14 and 15), recombined with linearized pPIC9K-3*B respectively to obtain pPIC9K-5*B, pPIC9K-6*B and pPIC9K-7*B, positive clones are screened by applying to LB plates containing Kan, plasmids of the screened positive clones are extracted, and the length of the plasmids is checked by enzyme cutting electrophoresis, so that the number of the repeated human-like collagen gene monomer contained in the plasmids contained in the obtained recombinant escherichia coli is judged.

Cloning the constructed tandem genes with proper repetition numbers into selected expression plasmids to obtain recombinant plasmids which can be used for transforming host cells for expression, and extracting plasmids to obtain a large amount of recombinant plasmids which can be used for transforming Pichia pastorisPichia Pastoris) Is a plasmid of (a).

Constructing a host strain Pichia pastoris for expressing collagen:

the successfully constructed pPIC9K-5*B, pPIC9K-6*B and pPIC9K-7*B plasmids were usedSacI linearizing it, after conversion of GS115, the methanol utilization flash (Mut ⁺ ) Recombinant yeast. By colony PCR, it was initially determined that linearized pPIC9K-5*B, pPIC9K-6*B and pPIC9K-7*B were homologously recombined into the chromosome of GS 115.

MD was electrotransfer coated and replica plated onto YPD plates containing varying concentrations of G418 to give multiple copies of recombinant yeast strain with an anti-G418 concentration of 4.00G/L. One strain is selected for expression experiment SDS-PAGE as shown in FIG. 3.

EXAMPLE 3 construction of Co-directed tandem fragments and strains of different genes

Collecting recombinant plasmids pPIC9K-B (i.e. containing plasmid with nucleotide sequence shown as SEQ ID NO. 2) and pPIC9K-C (i.e. containing plasmid with nucleotide sequence shown as SEQ ID NO. 4) and pPIC9K-E (i.e. containing plasmid with nucleotide sequence shown as SEQ ID NO. 6)By CaC1 ₂ Transforming Escherichia coli DH5 alpha by a method, screening by an LB plate containing Kan to obtain recombinant Escherichia coli containing plasmids pPIC9K-B, pPIC K-C and pPIC9K-E, namely DH5 alpha/pPIC 9K-B, DH5 alpha/pPIC 9K-C and DH5 alpha/pPIC 9K-E, culturing DH5 alpha/pPIC 9K-B, DH alpha/pPIC 9K-C and DH5 alpha/pPIC 9K-E in a liquid LB culture medium overnight, extracting plasmids to obtain a large number of pPIC9K-B, pPIC K-C and pPIC9K-E serving as templates, adding a homology arm of B at the tail end of C by using primers (R-C+B and F-C+B nucleotide sequences are shown as SEQ ID No.17 and 18), and then adding an E homology arm at the tail end of B by using a primer (R-B+E and F-B+E nucleotide sequences are shown as SEQ ID No.19 and 20), using a C fragment containing the B homology arm and B and E fragments of the E homology arm as templates, and carrying out PCR by using the primer (F-9K+C and the primer R-E+9K nucleotide sequences are shown as SEQ ID No.16 and 21) to obtain a large fragment by linking together the C+B+E, and cutting the gel to obtain a fragment (nucleotide sequence is shown as SEQ ID No. 9) of the C+B+E, wherein the amino acid sequence of the translated protein of the fragment C+B+E is shown as SEQ ID No. 8.

SEQ ID No.16 F-9K+C GAGGCTGAAGCTTACGTAgaattcGGTTTGAGAGGTGGAGC

SEQ ID No.17 R-C+B GAGGTCCACGAGCACCTTGTGGACCTGGTGGACCTC

SEQ ID No.18 F-C+B GAGGTCCACCAGGTCCACAAGGTGCTCGTGGACCTCCAG

SEQ ID No.19 R-B+E CCGTCATTACCTCTAGCACCTGGTGGACCTCTCTCGCC

SEQ ID No.20 F-B+E GGCGAGAGAGGTCCACCAGGTGCTAGAGGTAATGACGG

SEQ ID No.21 R-E+9K CGAATTAATTCGCGGCCGCctaTGGTTGACCTCTTGGAC

Recombining the C+B+E fragment to linearized pPIC 9K%EcoRI, restriction linearization) to obtain pPIC9K-C+B+E strain, and extracting plasmid to obtain pPIC9K-C+B+E, the structure is shown in figure 4.

The recombinant plasmid pPIC9K-C+B+E shown in FIG. 4 was passed throughSacI, respectively converting GS115 hosts after enzyme digestion linearization, screening by antibiotics to obtain corresponding engineering bacteria, fermenting and culturing (specific operation procedures are the same as those of example 1 and example 2), selecting one strain for expression experiment, and obtaining recombinant collagen with amino acid sequence shown in SEQ ID No.8 and polyacrylamideThe electrophoresis pattern, SDS-PAGE, is shown in FIG. 5.

Recombining the C+B+E fragment to linearized pPIC9K-C+B+E to obtain pPIC9K- (C+B+E) 2 (namely, a plasmid containing a nucleotide sequence shown as SEQ ID NO. 11), coating the plasmid on an LB plate containing Kan to screen positive clones, extracting the plasmid of the screened positive clones, carrying out enzyme digestion electrophoresis to check the length of the plasmid, and judging the repetition number of the human-like collagen gene monomer contained in the plasmid contained in the obtained recombinant escherichia coli.

Passing the recombinant plasmid pPIC9K- (C+B+E) x 2SacI, respectively converting GS115 hosts after enzyme digestion linearization, screening by antibiotics to obtain corresponding engineering bacteria, fermenting and culturing (the specific operation flow is the same as that of example 1 and example 2), selecting one strain for expression experiment, and obtaining the amino acid sequence of the recombinant human collagen shown as SEQ ID No.10, wherein a polyacrylamide electrophoresis diagram, namely SDS-PAGE, is shown as FIG. 6.

Example 4 fermentation and post-treatment Process of collagen Strain

(1) The fermentation culture conditions and recombinant collagen induction expression conditions of the genetically engineered bacteria are as follows:

adding Pichia pastoris cultured by the seed culture medium into a fermentation tank containing a batch fermentation medium according to 10% of inoculation amount to start culturing, regulating the stirring rotation speed to be 100 rpm-600 rpm, the tank pressure to be 0.03-0.07 MPa and the air flow to be 0.2 m ³ /h ~0.6 m ³ /h, dissolving oxygen>30％。

The formula of the seed culture medium is as follows: 100 mL phosphate buffer at pH 6.0, 1.34% YNB, 4X 10 ^-5 % biotin, 1% v/v glycerol.

Wherein the basic culture medium formula of the fermentation tank is as follows: 85% H ₃ PO ₄ 26.7 mL/L ;CaSO ₄ 0.93 g/L;K ₂ SO ₄ 18.2 g/L；MgSO ₄ •7H ₂ O14.9 g/L; KOH 4.13 and g/L; glycerol 40.0 g/L, and PTMI microelements are added after sterilization; wherein the PTMI microelements are as follows: cuSO ₄ •5H ₂ O 6.0 g/L；NaI 0 .08g/L；MnSO ₄ •H ₂ O 3.0 g/ L；Na ₂ MoO ₄ •2H ₂ O 0.2 g/L；H ₃ BO ₃ 0.02 g/L；CoCl 0.5g/L；ZnCl ₂ 20.0g/L；FeSO ₄ •7H ₂ O65.0 g/L; biotin 0.2 g/L; h ₂ SO ₄ 5.0 mL/L, filter sterilized with a 0.2 μm filter and stored at-4 ℃.

When the carbon source is exhausted, the dissolved oxygen is suddenly increased, the feeding of the feed supplement growth culture medium is started, the feeding rate is maintained to be more than 20 percent, the wet weight of the thalli reaches 180-220 g/L, and the feeding of the glycerol is stopped, wherein the formula of the feed supplement growth culture medium is as follows: 50% w/v glycerol containing 12 mL PTMI trace elements per liter;

after glycerol is exhausted, supplementing a fermentation induction culture medium for induction expression, and leading dissolved oxygen to be more than 20% by adjusting the rotating speed, the tank pressure, the air flow and the fed-batch methanol speed, and after induced fermentation for 96-120 hours, ending fermentation and collecting fermentation liquor, wherein the formula of the fermentation induction culture medium is as follows: 100% methanol, containing 12 mL PTMI microelements per liter.

(2) The recombinant collagen is extracted and purified as follows

And (3) centrifugally separating the fermentation liquor, collecting supernatant, filtering the supernatant by using a filter membrane, separating and purifying by using gel column chromatography, and freeze-drying to obtain a finished product. The specific purification steps of the humanized recombinant collagen are as follows:

centrifuging the fermentation broth at 6500 rpm for 20 min, collecting supernatant,

the supernatant was concentrated and washed with ultrafiltration membrane to remove salt and pigment, and the final conductance of the collagen solution was 1.77 ms/cm.

And (3) separating and purifying the collagen solution by using Unigel-80SP resin column chromatography of Nami company, collecting eluent containing collagen, washing the eluent by using an ultrafiltration membrane, desalting and concentrating, and finally conducting the electric conductivity of 0.48 ms/cm.

The target protein is collected by freeze drying of a freeze dryer, the purity is 98%, and an HPLC detection spectrogram is shown in figure 7.

EXAMPLE 5 comparison of the hydrophilicity of recombinant collagen with native collagen

The amino acid sequences of the recombinant human collagen and the natural human collagen are respectively submitted to a ProtScale server (https:// web. Expasy. Org/protscan /) for hydrophobic calculation, the calculation parameters adopt default Hphob/Kyte & Doolittle, the sliding window size is 9, a linear weighting model, the amino acid sequences of the recombinant human collagen and the natural human three-type alpha-chain collagen (GenBank accession number: AGL 34959.1) are subjected to hydrophobic analysis, the evaluation result is shown in figure 8, a blue histogram is the natural collagen, a red scattered area is the recombinant B collagen fragment designed by artificial combination, a purple broken line is the recombinant C collagen fragment designed by artificial combination, and a green broken line is the recombinant E collagen fragment designed by artificial combination. The lower the hydrophilicity-hydrophobicity rating, the better the hydrophilicity, i.e., the hydrophilicity of the recombinant B, C, E collagen fragment is significantly better than that of the native collagen.

The above examples are provided to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. Further, various modifications of the methods set forth herein, as well as variations of the methods of the invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art are intended to be within the scope of the present invention.

Claims (11)

1. Recombinant collagen, characterized in that it comprises one or more of the following features:

1) The amino acid sequence of the recombinant collagen is shown as SEQ ID No.1 or SEQ ID No. 8;

2) The recombinant collagen is protein which is derived from 1) and has collagen activity and is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence in 1).

2. The recombinant collagen according to claim 1, further comprising one or more of the following features:

i) The recombinant collagen is type III collagen;

II) the recombinant collagen is formed by serial connection of one or more repeated units with the amino acid sequence shown as SEQ ID No.1 or SEQ ID No. 8.

3. An isolated nucleic acid molecule encoding the recombinant collagen according to claim 1 or 2.

4. The nucleic acid molecule of claim 3, wherein the nucleotide sequence of the nucleic acid molecule is set forth in SEQ ID No.2 or SEQ ID No. 9.

5. An expression vector comprising the nucleic acid molecule of claim 3 or 4 and a plasmid backbone.

6. The expression vector of claim 5, wherein the plasmid backbone is selected from one or more of the group consisting of a pET expression vector, a pCW expression vector, a pUC expression vector, a pAO815 expression vector, a pPIC9K expression vector, a pPIC3.5K expression vector, a ppicza B expression vector, a ppicza C expression vector, a pGAPZ a expression vector, a ppiczb expression vector, a pPICZ C expression vector, a pGAPZ a expression vector, a pGAPZ B expression vector, or a pGAPZ C expression vector.

7. A host cell comprising the nucleic acid molecule of claim 3 or 4 or the expression vector of claim 5 or 6.

8. The host cell of claim 7, wherein the host cell is selected from the group consisting of a bacterial cell, a fungal cell, a plant cell, and an animal cell.

9. The cell of claim 8, wherein the bacteria are selected from the group consisting of escherichia coli, lactobacillus, bacillus licheniformis, bacillus subtilis, and streptomyces; or, the fungal cell is selected from Saccharomyces cerevisiae, candida, torulopsis, rhodotorula, hansenula, pichia or Kluyveromyces; or, the plant cell is selected from a tobacco cell or an arabidopsis cell; or, the animal cell is selected from Sf9 cell, CHO cell, NS0 cell, sp2/0 cell, HEK293 cell, HKB11 cell, per.c6 cell, heLa cell or CAP cell.

10. The method of preparing recombinant collagen according to claim 1 or 2, characterized in that the method of preparing comprises one or more of the following steps:

1) A host cell according to any one of claims 7-9 under suitable culture conditions;

2) Inducing the expression of the target gene of the host cell in step 1) to obtain recombinant collagen;

3) Harvesting host cells and/or culture medium containing recombinant collagen, and separating and purifying the recombinant collagen in the step 2).

11. Use of the recombinant collagen according to claim 1 or 2, the nucleic acid molecule according to claim 3 or 4, the expression vector according to claim 5 or 6 or the host cell according to any one of claims 7 to 9 for the preparation of a cosmetic, skin care or tissue material.