CN111334512B

CN111334512B - Recombinant human-like collagen containing hydroxyproline and hydroxylysine and production method thereof

Info

Publication number: CN111334512B
Application number: CN201911238876.9A
Authority: CN
Inventors: 黄亚东; 张齐; 肖巧学
Original assignee: Taiyuan Guangzhou Biotechnology Co ltd
Current assignee: Taiyuan Guangzhou Biotechnology Co ltd
Priority date: 2019-12-06
Filing date: 2019-12-06
Publication date: 2023-10-13
Anticipated expiration: 2039-12-06
Also published as: CN111334512A

Abstract

The invention discloses recombinant human-like collagen containing hydroxyproline and hydroxylysine and a production method thereof. The nucleotide sequence of the recombinant human-like collagen containing hydroxyproline and hydroxylysine is shown as SEQ ID NO. 1; the amino acid sequence of the recombinant human-like collagen containing hydroxyproline and hydroxylysine is shown as SEQ ID NO. 5. The recombinant human-like collagen is obtained by using Kluyveromyces as a chassis cell, respectively coexpression of four different vectors, namely pKLAC1-hlCOL, pKLC-P4HA, pKLC-PDI and pKLC-LH, and a series of fermentation and purification methods. The hydroxylation rate of the recombinant human-like collagen proline and lysine is similar to that of natural human-like collagen I, and reaches 30.7% and 43.1% respectively. Compared with non-hydroxylated recombinant human-like collagen, the collagen has better functions of promoting cell adhesion and promoting wound healing.

Description

Recombinant human-like collagen containing hydroxyproline and hydroxylysine and production method thereof

Technical Field

The invention relates to the technical field of biological recombinant proteins, in particular to recombinant human-like collagen containing hydroxyproline and hydroxylysine and a production method thereof.

Background

Collagen is the major structural protein of vertebrates and accounts for about 30% of the total protein of organisms. More than half of the extracellular matrix proteins of human tendons, bones, and skin are composed of collagen. Collagen, which is an important biological material with its special structure and function, has been used in the fields of drug delivery, surgical suturing, tissue filling, nerve repair, skin beauty, etc.

To date, the main sources of collagen are still animal-derived collagen, such as seafood like fish and livestock processing byproducts like cattle and pigs, which are obtained through extraction processes of acid, alkali, enzyme and the like, but the animal-derived collagen has the following obvious defects: 1) The final product has complex components and undefined active component structure, and the active structure is easy to be destroyed in the process of intense extraction; 2) The non-human protein has immunogenicity and the risk of anaphylactic reaction is not ignored.

Along with the development of biotechnology, the expression production of the collagen is realized by taking microorganisms as chassis cells, so that the inherent difficult problem of animal-derived collagen can be well solved. Firstly, humanization can greatly improve the biocompatibility, and solve the problem of immunogenicity; secondly, the structural design of the recombinant protein is stronger in purpose, the components are more definite, and the biological activity of the collagen can be greatly improved. Based on this, research and application of recombinant collagen have been in an increasing trend in recent years.

Chinese patent application CN201910091891.9 discloses a method for producing recombinant human type II collagen single chain by using Pichia pastoris. PCT/US2018/053601 discloses a method for expressing recombinant human-like collagen using a variety of microbial expression systems. Although the method has certain benefits, the structural design of the recombinant collagen is still not clear, especially the loss of hydroxylation functions of proline and lysine restricts the further application of the recombinant collagen.

The natural collagen has complex structure and large molecular weight, and compared with the collagen extracted from animal sources, the recombinant collagen is still difficult to 'win the peptides' at present, and the 'win the peptides' can be better realized only by designing a more reasonable and effective structure. In addition, the natural collagen structure contains about 23% hydroxyproline and hydroxylysine. These hydroxylation sites are closely related to collagen structure formation, fibril function, tissue development, and a variety of pathologies. For example, lack of hydroxylysine can cause diseases such as Ehlers-Danlos syndrome type VI, bruck Syndrome (BS), and skeletal dysplasia. With age, the content of lysine hydroxylase in human skin tissue appears to decrease drastically.

Christoph R et al achieved hydroxylation of recombinant collagen in E.coli (Recombinant expression of hydroxylated human collagen in Escherichia coli, 2013); chinese patent application CN201810707153.8 discloses a method for implementing proline hydroxylation of collagen by co-expressing proline hydroxylase derived from cystosporium RH1 and a human collagen gene in escherichia coli. Although such methods solve the problem of partial hydroxylation of recombinant collagen to some extent, the following disadvantages also exist: 1) The structural function design of the recombinant human collagen is still unclear; 2) For E.coli with complete hydroxylation, E.coli belongs to a non-GRAS expression host (Generally Recognized as Safe, the safety index of food additives evaluated by the FDA in the United states), and in addition, the downstream purification process is complex and the endotoxin removal difficulty is high. 3) For other expression hosts, hydroxylation is limited to proline hydroxylation, and synchronous hydroxylation of proline and lysine is difficult to achieve.

Disclosure of Invention

In view of the above-mentioned shortcomings of the prior art, the present invention aims to provide a recombinant human-like collagen containing hydroxyproline and hydroxylysine while the hydroxylation host is a GRAS expression host, which has definite structural domain active site and realizes synchronous hydroxylation of proline and lysine, and a production method thereof.

The invention firstly designs and combines the amino acid sequences of the human-like collagen, and endows the collagen with more compact structure and higher biological activity. Then constructing a plurality of expression vectors by using a genetic engineering method, and successfully introducing the hydroxylated exogenous genes and the human-like collagen genes into the Kluyveromyces. Finally, the production of the hydroxylation human-like collagen is realized by taking galactose as an inducer through a microbial fermentation technology.

In order to achieve the above purpose, the invention adopts the following technical scheme:

nucleotide sequence of recombinant human-like collagen containing hydroxyproline and hydroxylysine: the nucleotide sequence is shown as SEQ ID NO. 1.

Recombinant human-like collagen containing hydroxyproline and hydroxylysine: the amino acid sequence of the recombinant human-like collagen containing hydroxyproline and hydroxylysine is shown as SEQ ID NO. 5.

Kluyveromyces lactis (Kluyveromyces lactis) expressing recombinant human-like collagen: the food-grade expression host Kluyveromyces is taken as a chassis cell, and secretion expression of the hydroxylation recombinant human-like collagen is realized by co-transforming expression vectors containing proline hydroxylase alpha subunit, protein disulfide isomerase, lysine hydroxylase L230 and the recombinant human-like collagen; the hydroxylation recombinant human-like collagen is recombinant human-like collagen containing hydroxyproline and hydroxylysine; wherein the proline hydroxylase alpha subunit, the protease disulfide isomerase and the lysine hydroxylase are expressed in an intracellular constitutive mode, and the recombinant human-like collagen is expressed in a secretory mode; the nucleotide sequence of the alpha subunit of the proline hydroxylase is shown as SEQ ID No.2, and the amino acid sequence is shown as SEQ ID No. 6; the nucleotide sequence of the protease disulfide isomerase is shown as SEQ ID No.3, and the amino acid sequence is shown as SEQ ID No. 7; the nucleotide sequence of lysine hydroxylase is shown as SEQ ID No.4, and the amino acid sequence is shown as SEQ ID No. 8; kluyveromyces lactis (Kluyveromyces lactis) expressing recombinant human-like collagen has a accession number of: GDMCC NO 60895. The preservation address is: guangzhou city, first, the middle road 100 # college 59 # building 5, the post code is 510070; preservation unit: the cantonese institute for microorganisms; the preservation time is 2019, 11, 15.

The production induction method of kluyveromyces expressing recombinant human-like collagen comprises the following steps:

1) Inoculating: selecting single colony of Kluyveromyces in YPD medium, culturing at 28-30deg.C and 100-300rpm for 16-30 hr to OD ₆₀₀ Reaching 20-30;

2) Fermentation culture: inoculating the seed liquid obtained in the step 1) into a fermentation tank, controlling the temperature to be 28-30 ℃, controlling the dissolved oxygen to be not lower than 20%, and controlling the pH to be 5.8+/-0.1;

3) And (3) growing thalli: after the glucose is exhausted, glucose is added;

4) Galactose induction: after the glucose in the step 3) is consumed, starting to supplement galactose from a plant source until the induction is finished;

5) Centrifuging, and collecting the supernatant of the fermentation broth to obtain the recombinant human-like collagen fermentation broth.

Preferably, step 3) is supplemented with 0.1% -12% glucose.

Preferably, step 4) is supplemented with 0.1% -10% galactose of vegetable origin

Preferably, the centrifugation in step 5) is carried out for 2-30min at 3000g-10000 g.

The recombinant human-like collagen containing hydroxyproline and hydroxylysine is applied to promoting cell adhesion and wound healing.

Compared with the prior art, the invention has the following advantages and beneficial effects:

1) The invention takes food-grade expression host Kluyveromyces as chassis cells, successfully realizes secretory expression of hydroxylation recombinant human-like collagen by coexpression of alpha subunit of humanized proline hydroxylase (P4H), protein Disulfide Isomerase (PDI), lysine hydroxylase L230 and human-like collagen, and solves the secretory expression and hydroxylation problems of recombinant human-like collagen by respectively reaching 30.7 percent and 43.1 percent of hydroxylation rates of proline and lysine.

2) The recombinant human-like collagen obtained by the invention has definite structural domain, has hydroxylation rate of proline and lysine similar to that of natural collagen, and has better cell proliferation and adhesion promoting activity.

3) The chassis cell is GRAS expression host, and realizes the secretion expression of recombinant collagen, simplifying the production process.

4) The recombinant human-like collagen obtained by the invention has single component and high purity, and does not contain endotoxin.

5) The recombinant human-like collagen obtained by the invention can be used for promoting the repair of epidermal cells, accelerating the hemostatic healing of wounds, strengthening skin barriers, serving as a tissue filler and other pharmaceutical or cosmetic fields.

Drawings

FIG. 1 shows the physical map of the pKLAC1-hlCOL plasmid.

FIG. 2 shows the physical map of pKLC1-P4HA plasmid.

FIG. 3 is a physical map of pKLC1-LH plasmid.

FIG. 4 is a physical map of the pKLC1-PDI plasmid.

FIG. 5 shows SDS-PAGE of positive recombinant-induced supernatants.

FIG. 6 is a graph showing the change in the content of proline and hydroxylysine in the supernatant of the fermentation in the fermenter of example 4.

FIG. 7 is a HPLC purity detection peak diagram for hydroxylated recombinant human-like collagen of example 5.

FIG. 8 shows the adhesive activity (n=6) of recombinant human-like collagen samples at various concentrations to NIH/3T3 cells of example 6.

FIG. 9 is a graph of wound repair process (scale: 5 mm) at various time points of a rat full skin defect model.

Detailed Description

The present invention will be further described with reference to examples and drawings for the purpose of making the objects, technical solutions and effects of the present invention clearer and more specific, but the embodiments of the present invention are not limited thereto.

Example 1: recombinant human-like collagen nucleotide and amino acid sequence design, construction of expression vector

Recombinant human-like collagen (hlCOL), alpha subunit of proline hydroxylase (P4H), protein Disulfide Isomerase (PDI), and protein genes hlCOL and P4HA, PDI, LH of lysine hydroxylase L230 (the code of lysine hydroxylase L230 is LH) are respectively designed through an online website (http:// www.jcat.de /). The nucleotide sequence of the synthesized recombinant human-like collagen (hlCOL) is shown as SEQ ID No.1, the nucleotide sequence of alpha subunit of proline hydroxylase (P4H) is shown as SEQ ID No.2, the nucleotide sequence of lysine hydroxylase is shown as SEQ ID No.3 and the nucleotide sequence of the alpha subunit of proline hydroxylase (P4H) is shown as SEQ ID No. 4. The amino acid sequences obtained by coding and expressing the nucleotide sequences of recombinant human-like collagen (hlCOL), alpha subunit of proline hydroxylase (P4H), protease Disulfide Isomerase (PDI) and lysine hydroxylase L230 are shown as SEQ ID No.5, SEQ ID No.6, SEQ ID No.7 and SEQ ID No.8 respectively. The gene sequences and amino acid sequences corresponding to recombinant human-like collagen (hlCOL), alpha subunit of proline hydroxylase (P4H), protein Disulfide Isomerase (PDI), lysine hydroxylase L230 (lysine hydroxylase L230 is labeled LH) and human-like collagen are shown in table 1.

TABLE 1 Gene sequences and corresponding amino acid sequences according to the invention

The upstream primer F and the downstream primer R (assigned to Shanghai bioengineering Co., ltd.) corresponding to the recombinant human-like collagen (hlCOL), alpha subunit of proline hydroxylase (P4H), protein Disulfide Isomerase (PDI), and protein (LH) genes of lysine hydroxylase L230 were synthesized, respectively, as shown in Table 2; the sequences of an upstream primer hlCOL-F and a downstream primer hlCOL-R of hlCOL are respectively shown as SEQ ID NO.9 and SEQ ID NO. 10; the sequences of the upstream primer P4H-F and the downstream primer P4H-R of the P4H are respectively shown as SEQ ID NO.11 and SEQ ID NO. 12; the sequences of the upstream primer PDI-F and the downstream primer PDI-R of the PDI are respectively shown as SEQ ID NO.13 and SEQ ID NO. 14; the sequences of the upstream primer LH-F and the downstream primer LH-R of LH are respectively shown as SEQ ID NO.15 and SEQ ID NO. 16; in Table 2, F corresponds to the upstream primer and R corresponds to the downstream primer. The corresponding gene fragment was amplified by PCR and the corresponding cleavage sites HindIII (CTCGAG), notI (GCGGCCGC) and XhoI (AAGCTT) were introduced. The PCR reaction system is shown in Table 3.

TABLE 2

TABLE 3PCR reaction System

The pKALC1 vector is taken as a framework, the synthesized gene fragments are respectively introduced into the vector through HindIII-NotI cleavage sites and XhoI-NotI cleavage sites, and the constructed novel vector is respectively named pKLAC1-hlCOL (plasmid A, corresponding physical map is shown in figure 1), pKLC-P4HA (plasmid B, corresponding physical map is shown in figure 2), pKLC-PDI (plasmid C, corresponding physical map is shown in figure 3) and pKLC-LH (plasmid D, corresponding physical map is shown in figure 4). Wherein, plasmid A is a secretion expression vector, secretion expression human-like collagen, plasmid B, plasmid C and plasmid D are intracellular expression vectors, the combination of the subunits of the expression proteins of plasmid B and plasmid C has proline hydroxylase activity, and the protein expressed by plasmid D has lysine hydroxylase activity.

The ligation product was transformed into E.coli JM109 competent by heat shock at 42℃and, after resuscitating, was plated on ampicillin-resistant (100. Mu.g/mL) LB-resistant plates, single colonies were picked overnight at 37℃and PCR verified using the corresponding primers in Table 3 (reaction system is as in Table 3). Positive transformants were selected for sequencing.

Example 2: construction of hydroxylation collagen-like engineering bacteria

As shown in FIGS. 1, 2, 3 and 4, plasmid A, plasmid B, plasmid C and plasmid D were linearized with restriction enzyme SacII (NEB Co. USA) to construct the cleavage reaction system as shown in Table 4 below:

TABLE 4 cleavage reaction System

The reaction conditions were 37℃for 30min. After the enzyme digestion reaction is finished, the carrier fragment containing the target gene is recovered by utilizing a rubber recovery kit (Beijing Tiangen organism) rubber cutting, and the resistance gene fragment on the carrier is removed.

The recovered high purity plasmid fragment was added to Kluyveromyces competent cells, placed on ice for incubation for 2min, and subjected to electrotransformation (1.5 kv-2.5 kv). Then, the cells were plated on YCB medium containing acetamide (5 mM) and cultured at 28℃for 2 days, and after single colony was grown on the plate, kluyveromyces lactis (Kluyveromyces lactis) expressing recombinant human-like collagen was obtained with a accession number of: GDMCC NO 60895. The preservation address is: guangzhou city, first, the middle road 100 # college 59 # building 5, the post code is 510070; preservation unit: the cantonese institute for microorganisms; the preservation time is 2019, 11, 15.

Single colonies were picked and verified by colony PCR. In this example, corresponding upstream and downstream primers were designed according to the target gene, respectively, as shown in Table 5 (the synthesis of Shanghai Biotechnology Co., ltd., F 'represents the upstream primer, and R' represents the downstream primer).

TABLE 5

To the PCR cuvette, 25. Mu.L of a bacterial lysate (Takara Co.) was added, and different transformants were picked up, placed in the PCR cuvette, and then removed after blowing. The PCR vials were then denatured at 80℃for 15min, centrifuged at 2000g for 1min, and 3. Mu.L of supernatant was used as template for the PCR reaction. The PCR reaction system is shown in Table 6 below:

TABLE 6 colony PCR reaction System

And (3) carrying out nucleic acid electrophoresis on the PCR product, analyzing the size of the band, and carrying out the next step of identification when the actual value is consistent with the theoretical value.

The positive transformants were cultured, and the fermentation supernatants induced for 24 hours were subjected to polyacrylamide gel electrophoresis (SDS-PAGE), and the results are shown in FIG. 5. Meanwhile, the content of hydroxyproline and the content of hydroxylysine are measured.

When the results were positive, the bands of the electrophoresis gel which were the same as the theory were cut off, and protein gel analysis (completed by Shanghai Ming biosciences Co., ltd.) was performed, and the amino acid composition results were shown in Table 7. The amino acid composition accords with the theory, and the hydroxylation rates of the proline and the lysine respectively reach 30.7 percent and 43.1 percent, which are similar to those of the natural human type I collagen.

Analysis of amino acid composition by human collagen gel strip

Example 3: hydroxyproline and hydroxylysine content determination

Hydroxyproline was measured spectrophotometrically in the Ministry of agriculture industry Standard, determination of L-hydroxyproline in raw milk (NY/T3130-2017).

Hydroxylysine was detected using a hydroxylysine ELISA detection kit (available from Shanghai-Ming Biotech Co.). The method comprises the following steps: 10. Mu.L of each diluted standard (kit self) or fermentation supernatant (source example 2) was taken and 40. Mu.L of the diluent was added; blank holes are not added. In addition to the blank wells, 100. Mu.L of horseradish peroxidase (HRP) -labeled detection antibody was added to each of the standard wells and sample wells, and the reaction wells were sealed with a sealing plate membrane and incubated for 60min in a 37℃water bath or incubator. Removing liquid, beating to dry on the water-absorbing paper, filling the washing liquid in each hole, standing for 1min, throwing away the washing liquid, beating to dry on the water-absorbing paper, and repeating the plate washing for 5 times. Substrate A, B was added 50. Mu.L each to each well and incubated at 37℃for 15min in the absence of light. Then, 50. Mu.L of a stop solution was added thereto for 15 minutes, and the OD of each well was measured at a wavelength of 450 nm. Drawing a standard curve according to the result: in an Excel worksheet, standard substance concentration is taken as an abscissa, a corresponding OD value is taken as an ordinate, a standard substance linear regression curve is drawn, and each sample concentration value is calculated according to a curve equation.

Example 4: induction production process of recombinant human-like collagen on 5L fermentation tank

First, the kluyveromyces lactis (Kluyveromyces lactis) engineering bacteria expressing recombinant human-like collagen constructed in example 2 were strain-activated. The YPD solid plate was streaked with the inoculum solution dipped in the inoculating loop. Culturing at 30deg.C for 2 days, picking single colony in YPD medium, culturing at 30deg.C and 200rpm for about 30 hr to OD ₆₀₀ And the seed liquid reaches 20-30, namely the first-stage seed liquid, and the seed liquid can be subjected to expansion culture according to the final culture volume size in a ratio of 1:10.

Inoculating the prepared seed solution into a 5L fermentation tank (CBM culture medium, initial volume of 2.4L) in a sterile inoculation mode, wherein the fermentation conditions are as follows: the temperature is 28 ℃, the dissolved oxygen is not lower than 20 percent, and the pH is controlled to be 5.8 plus or minus 0.1 by adding 25 percent ammonia water. After the glucose is exhausted, 5% -12% (w/v, the volume is the total fermentation volume) of sterilized glucose is continuously added; after glucose supplementation is completed, supplementation of 2% -5% (w/v, volume is total fermentation volume) of plant-derived beta-galactose (filtered sterilization, purchased from Zhejiang new pharmaceutical Co., ltd.) is started until induction is completed.

Wherein, the formula of the fermentation medium is as follows:

CBM culture medium (4 mL trace element per liter basal medium)

Basal medium: 85% phosphoric acid 28mL/L, calcium sulfate 1g/L, potassium sulfate 18g/L, magnesium sulfate heptahydrate 14g/L, potassium hydroxide 4g/L, yeast extract 1%, glucose 2% (separately sterilized).

Trace elements: 6.0g/L of copper sulfate pentahydrate, 0.008g/L of sodium iodide, 3.0g/L of manganese sulfate monohydrate, 0.2g/L of sodium molybdate dihydrate, 0.02g/L of boric acid, 0.5g/L of cobalt chloride, 20.0g/L of zinc chloride, 65.0g/L of ferrous sulfate heptahydrate, 0.2g/L of biotin and 5mL/L of concentrated sulfuric acid are subjected to centrifugation for 10min after fermentation, and the supernatant of the fermentation broth is collected to be the recombinant human-like collagen fermentation broth. The change of hydroxyproline and hydroxylysine content during the whole fermentation induction process is shown in figure 6. FIG. 6 shows that hydroxyproline and hydroxylysine in the supernatant of the fermentation broth begin to accumulate rapidly after 24h of induction, and the analytical reasons may be related to the intracellular activities of the introduced hydroxyproline and hydroxylysine, indicating that hydroxyproline and hydroxylysine can accumulate stably during production.

Example 5: purification of high-purity recombinant human-like collagen

The hydroxylated recombinant human-like collagen fermentation broth collected by centrifugation is incubated with nickel filler (Changzhou heaven and earth) for 5min in a volume ratio of 50:1, and then the nickel filler is continuously added until the protein content in the supernatant is no longer reduced.

The packing was collected by filtration, then washed with 5 volumes of 0.1M PBS, then washed with 0.5M imidazole, the wash was collected, and imidazole was replaced with 0.1M PBS via a G25 molecular sieve (american GE). The purified protein was tested by HPLC (HPLC conditions: agilent 1260 high performance liquid chromatograph (diode array detector; equipped), chromatographic column: reverse phase C18 column, hedera ODS-2.6 x 250.5um. Mobile phase: A0.1% TFC water, B0.1% TFC acetonitrile, gradient elution: 0-40 min, 1% B-70% B, detection wavelength 215 nm), and purity as high as 99.138% as shown in FIG. 7.

Example 6: recombinant human-like collagen activity assay

3T3 cells in logarithmic growth phase were routinely cultured to 80% -90% with 1640 (Gibco Co., USA) containing 10% FBS, digested with 0.25% trypsin, and 2.0X10 ⁵ The cell number of each/mL is inoculated in a 96-well plate, after 24 hours of culture, the culture medium is replaced by a serum-free 1640 culture medium, after 24 hours, the culture medium is sucked out, recombinant human-like collagen with the concentration of 100, 50, 25 and 10 mug/mL is respectively added (all samples are diluted by PBS, and the control bovine fibronectin is 100 mug/mL), and 3 holes are arranged for calculating the error of each concentration. After administration, the mixture was cultured in a carbon dioxide incubator for 24 hours, 10. Mu.L of MTT (0.5 mg/mL) was added,after incubation for 4h in an incubator at 37℃the solution in the wells was aspirated and 100. Mu. LDMSO was added and the absorbance was measured at 570/630nm after shaking.

The results are shown in FIG. 8, and compared with bovine fibronectin, the recombinant human-like collagen has better cell adhesion promoting activity as compared with bovine fibronectin by 4 times under the condition of the same adding amount of 100 mug/mL.

Example 7: rat full-skin defect model repair

Healthy female clean-grade SD rats were taken in 96 groups of 16 randomly. Group A (PBS blank), group B (EGF control), group C (non-hydroxylated recombinant collagen), group D (100 ng hydroxylated recombinant collagen), group E (500 ng hydroxylated recombinant collagen), group F (1 μg hydroxylated recombinant collagen).

A circular wound surface is formed by taking a full layer of skin on the back of a rat by adopting a conventional operation method, the diameter of the wound surface is 2.5cm, and after the wound surface is hemostatic, the wound surface is covered with normal saline gauze for standby, and an animal full layer skin defect model is established. According to the above group, spray administration was performed on the wound surface (e.g., blank control group corresponding to the above A-F group, EGF control group and different types of collagen). The animals are routinely kept in single cages after operation, and are dosed once every 48 hours. The medicine is administered for 5-14 days respectively. From fig. 9, the wound healing situation of the hydroxylated recombinant human-like collagen is significantly better than that of the normal saline group (negative control) and the normal human-like collagen. It has the effects of accelerating wound healing and reducing scar formation.

Sequence listing

<110> peptide Source (Guangzhou) Biotechnology Co., ltd

<120> recombinant human-like collagen containing hydroxyproline and hydroxylysine and method for producing the same

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caatacagag tttctaagtc tgcttggttg tctggttacg aaaacccagt tgtttctaga 1140

atcaacatga gaatccaaga cttgactggt ttggacgttt ctactgctga agaattgcaa 1200

gttgctaact acggtgttgg tggtcaatac gaaccacact tcgacttcgc tagaaaggac 1260

gaaccagacg ctttcaagga attgggtact ggtaacagaa tcgctacttg gttgttctac 1320

atgtctgacg tttctgctgg tggtgctact gttttcccag aagttggcgc gtctgtatgg 1380

ccaaaaaagg gtactgctgt tttctggtac aacttgttcg cttctggtga aggtgactac 1440

tctactagac acgctgcttg cccagtcctc gttggcaata agtgggtaag caacaagtgg 1500

ttgcacgaaa gaggtcaaga attcagaaga ccatgtactt tgtctgaatt ggaataa 1557

<210> 3

<211> 1479

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

atggacgctc cagaagaaga agaccacgtt ttggttttga gaaagtctaa ctttgcggaa 60

gctctcgctg ctcacaagta cttgttggtt gaattctacg ctccatggtg tggtcactgt 120

aaggctttgg ctccagaata cgctaaggct gctggtaagt tgaaggctga aggttctgaa 180

atcagattgg ctaaggttga cgctactgaa gaatctgact tggctcaaca atacggtgtt 240

agaggttacc caactatcaa gttcttcaga aacggtgaca ctgcttctcc aaaggaatac 300

actgctggta gagaagctga cgacatcgtt aactggttga agaagagaac tggtccagct 360

gctactactt tgccagacgg tgctgctgct gaatctttgg ttgaatcttc tgaagttgct 420

gttatcggtt tcttcaagga cgttgaatct gactctgcta agcaattctt gcaagctgct 480

gaagctatcg acgacatccc attcggtatc acttctaact ctgacgtttt ctctaagtac 540

caattggaca aggacggtgt tgttttgttc aagaagttcg acgaaggtag aaacaacttc 600

gaaggtgaag ttactaagga aaacttgttg gacttcatca agcacaacca attgccattg 660

gttatcgaat tcactgaaca aactgctcca aagatcttcg gtggtgaaat caagactcac 720

atcttgttgt tcttgccaaa gtctgtttct gactacgacg gtaagttgtc taacttcaag 780

actgctgctg aatctttcaa gggtaagatc ttgttcatct tcatcgactc tgaccacact 840

gacaaccaaa gaatactcga attcttcgga ctcaagaagg aagagtgtcc agctgttaga 900

ttgatcactt tggaagaaga aatgactaag tacaagccag aatctgaaga attgactgct 960

gaaagaatca ctgaattctg tcacagattc ttggaaggta agatcaagcc acacttgatg 1020

tctcaagaat tgccagaaga ctgggacaag caaccagtta aggttttggt tggtaagaac 1080

ttcgaagacg ttgctttcga cgaaaagaag aacgttttcg ttgaattcta cgctccatgg 1140

tgtggtcact gtaagcaatt ggctccaatc tgggacaagt tgggtgaaac ttacaaggac 1200

cacgaaaaca tcgttatcgc taagatggac tctactgcta acgaagttga agctgttaag 1260

gttcactctt tcccaacttt gaagttcttc ccagcttctg ctgacagaac tgttatcgac 1320

tacaacggtg aaagaacttt ggacggtttc aagaagttct tggaatctgg tggtcaagac 1380

ggtgctggtg acgacgacga cttggaagac ttggaagaag ctgaagaacc agacatggaa 1440

gaagacgacg accaaaaggc tgttaaggac gaattgtaa 1479

<210> 4

<211> 2688

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

atgatctcta gaacttacgt tatcaacttg gctagaagac cagacaagaa ggacagaatc 60

ttggctgaat tcttgaagtt gaaggaaaag ggtgttgaat tgaactgtgt tatcttcgaa 120

gctgttgacg gtaacaaccc agaacacttg tctagattca acttcaagat cccaaactgg 180

actgacttga actctggtaa gccaatgact aacggtgaag ttggttgtgc tttgtctcac 240

tggtctgttt ggaaggacgt tgttgactgt gttgagaatg gtactctcga caaggactgc 300

agaatcttgg ttttggaaga cgacgttgtt ttcttggaca acttcatgga aagataccaa 360

acttacactt ctgaaatcac ttacaactgt gacttgttgt acttgcacag aaagccattg 420

aacccataca ctgaaactaa gatctctact cacatcgtta agccaaacaa gtcttactgg 480

gcttgtgctt acgttatcac ttaccaatgt gctaagaagt tcatgaacgc taactacttg 540

gaaaacttga tcccatctga cgaattcatc ccaatcatgc acggttgcaa tgtttacggc 600

ttcgaaaagt tgttttctaa ctgtgaaaag atcgactgtt acgctgttca accatctttg 660

gttaagttga cttctaacgc tttcaacgac tctgaaactt tccactctgg ttcttacgtt 720

ccatctaaca agttcaactt cgacactgac aagcaattca gaatcgttta catcggtcca 780

actaagggta actctttcca cagattcact gaatactgta agttgtactt gttgccatac 840

aaggttatcg acgaaaagga aactaacgac tttgttagct tgagatctga actccaatct 900

ttgtctgaac aagacttgaa cactactttg atgttggttg tttctgttaa ccacaacgac 960

ttctgtaaca ctatcccatg tgctccaact aacgaattca tcgacaagta caagcaattg 1020

actactgaca ctaactctat cgtttctgct gttcaaaacg gtactaacaa gactatgttc 1080

atcggttggg ctaacaagat ctctgaattc atcaaccact accaccaaaa gttgactgaa 1140

tctaacgctg aaactgacat caacttggct aacttgttgt tgatctcttc tatctcttct 1200

gacttcaact gtgttgttga agacgttgaa ggtaacttgt tccaattgat caacgaagaa 1260

tctgacatcg ttttctctac tactacttct agagttaaca acaagttggg taagactcca 1320

tctgttttgt acgctaactc tgactcttct gttatcgttt tgaacaaggt tgaaaactac 1380

actggttacg gttggaacga atactacggt taccacgttt acccagttaa gttcgacgtt 1440

ttgccaaaga tctacttgtc tatcagaatc gttaagaacg ctaacgttac taagatcgct 1500

gaaactttgg actacccaaa ggaattgatc actgtttcta tctctagatc tgaacacgac 1560

tctttctacc aagctgacat ccaaaagttc ttgttgtctg gtgctgacta ctacttctac 1620

atctctggtg actgtatcat cactagacca actatcttga aggaattgtt ggaattgaac 1680

aaggacttcg ttggtccatt gatgagaaag ggtactgaat cttggactaa ctactggggt 1740

gacatcgacc catctaacgg ttactacaag agatctttcg actacttcga catcatcggt 1800

agagacagag ttggttgttg gaacgttcca tacttggctt ctgtttactt gatcaagaag 1860

tctgttatcg aacaagttcc aaacttgttc actgaaaact ctcacatgtg gaacggttct 1920

aacatcgaca tgagattgtg tcacaacttg agaaagaaca acgttttcat gtacttgtct 1980

aacttgagac catacggtca catcgacgac tctatcaact tggaagtttt gtctggtgtt 2040

ccaactgaag ttactttgta cgacttgcca actagaaagg aagaatggga aaagaagtac 2100

ttgcacccag aattcttgtc tcacttgcaa aacttcaagg acttcgacta cactgaaatc 2160

tgtaacgacg tttactcttt cccattgttc actccagctt tctgtaagga agttatcgaa 2220

gttatggaca aggctaactt gtggtctaag ggtggtgact cttacttcga cccaagaatc 2280

ggtggtgttg aatcttaccc aactcaagac actcaattgt acgaagttgg tttggacaag 2340

caatggcact acgttgtttt caactacgtt gctccattcg ttagacactt gtacaacaac 2400

tacaagacta aggacatcaa cttggctttc gttgttaagt acgacatgga aagacaatct 2460

gaattggctc cacaccacga ctcttctact tacactttga acatcgcttt gaacgaatac 2520

ggtaaggaat acactgctgg tggttgtgaa ttcatcagac acaagttcat ctggcaaggt 2580

caaaaggttg gttacgctac tatccacgct ggtaagttgt tggcttacca cagagctttg 2640

ccaatcactt ctggtaagag atacatcttg gtttctttcg ttaactaa 2688

<210> 5

<211> 587

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 5

Gly Val Pro Gly Asp Leu Gly Ala Pro Gly Pro Ser Gly Ala Arg Gly

1 5 10 15

Glu Arg Gly Phe Pro Gly Glu Arg Gly Val Gln Gly Pro Pro Gly Pro

20 25 30

Ala Gly Pro Arg Gly Ala Asn Gly Ala Pro Gly Asn Asp Gly Ala Lys

35 40 45

Gly Asp Ala Gly Ala Pro Gly Ala Pro Gly Leu His Gly Glu Phe Gly

50 55 60

Leu Pro Gly Pro Ala Gly Pro Arg Gly Glu Arg Gly Pro Pro Gly Glu

65 70 75 80

Ser Gly Ala Ala Gly Pro Thr Gly Pro Ile Gly Ser Arg Gly Pro Ser

85 90 95

Gly Pro Pro Gly Pro Asp Gly Asn Lys Gly Glu Pro Gly Val Val Gly

100 105 110

Ala Val Pro Gly Pro Ala Gly Lys Glu Gly Gly Lys Gly Pro Arg Gly

115 120 125

Glu Thr Gly Pro Ala Gly Arg Pro Gly Glu Val Gly Pro Pro Gly Pro

130 135 140

Pro Gly Pro Ala Gly Glu Lys Gly Ser Pro Gly Ala Asp Gly Pro Ala

145 150 155 160

Gly Ala Pro Gly Thr Pro Gly Pro Gln Gly Ile Ala Gly Gln Arg Gly

165 170 175

Val Val Gly Leu Pro Pro Gly Pro Ala Gly Lys Glu Gly Leu Arg Gly

180 185 190

Pro Arg Gly Asp Gln Gly Pro Val Gly Arg Thr Gly Glu Val Gly Ala

195 200 205

Val Gly Pro Pro Gly Phe Ala Gly Glu Lys Gly Pro Ser Gly Glu Ala

210 215 220

Gly Thr Ala Gly Pro Pro Gly Thr Pro Gly Pro Gln Gly Leu Leu Gly

225 230 235 240

Ala Pro Gly Ile Leu Gly Leu Pro Gly Leu Pro Gly Gln Arg Gly Glu

245 250 255

Arg Gly Phe Pro Gly Leu Pro Gly Pro Ser Gly Glu Pro Gly Lys Gln

260 265 270

Gly Pro Ser Gly Ala Ser Gly Glu Arg Gly Pro Pro Gly Pro Met Gly

275 280 285

Pro Pro Gly Leu Ala Gly Pro Pro Gly Glu Ser Gly Arg Glu Gly Ala

290 295 300

Pro Gly Ala Glu Gly Ser Pro Gly Arg Asp Gly Ser Pro Gly Ala Lys

305 310 315 320

Gly Asp Arg Gly Glu Thr Gly Pro Ala Gly Pro Pro Gly Ala Pro Gly

325 330 335

Ala Pro Gly Ala Pro Gly Pro Val Gly Pro Ala Gly Lys Ser Gly Asp

340 345 350

Arg Gly Glu Thr Gly Pro Ala Gly Pro Thr Gly Pro Val Gly Pro Val

355 360 365

Gly Ala Arg Gly Pro Ala Gly Pro Gln Gly Pro Arg Gly Asp Lys Gly

370 375 380

Glu Thr Gly Glu Gln Gly Asp Arg Gly Ile Lys Gly His Arg Gly Phe

385 390 395 400

Ser Gly Leu Gln Gly Pro Pro Gly Pro Pro Gly Ser Pro Gly Glu Gln

405 410 415

Gly Pro Ser Gly Ala Ser Gly Pro Ala Gly Pro Arg Gly Pro Pro Gly

420 425 430

Ser Ala Gly Ala Pro Gly Lys Asp Gly Leu Asn Gly Leu Pro Gly Pro

435 440 445

Ile Gly Pro Pro Gly Pro Arg Gly Arg Thr Gly Asp Ala Gly Pro Val

450 455 460

Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly

465 470 475 480

Glu Arg Gly Asp Leu Gly Pro Gln Gly Ile Ala Gly Gln Arg Gly Val

485 490 495

Val Gly Glu Arg Gly Glu Arg Gly Glu Arg Gly Ala Ser Gly Glu Arg

500 505 510

Gly Asp Leu Gly Pro Gln Gly Ile Ala Gly Gln Arg Gly Val Val Gly

515 520 525

Glu Arg Gly Glu Arg Gly Glu Arg Gly Ala Ser Gly Glu Arg Gly Asp

530 535 540

Leu Gly Pro Gln Gly Ile Ala Gly Gln Arg Gly Val Val Gly Glu Arg

545 550 555 560

Gly Glu Arg Gly Glu Arg Gly Ala Ser Gly Pro Pro Gly Pro Cys Cys

565 570 575

Gly Gly Gly Leu Glu His His His His His His

580 585

<210> 6

<211> 587

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 6

Gly Val Pro Gly Asp Leu Gly Ala Pro Gly Pro Ser Gly Ala Arg Gly

1 5 10 15

Glu Arg Gly Phe Pro Gly Glu Arg Gly Val Gln Gly Pro Pro Gly Pro

20 25 30

Ala Gly Pro Arg Gly Ala Asn Gly Ala Pro Gly Asn Asp Gly Ala Lys

35 40 45

Gly Asp Ala Gly Ala Pro Gly Ala Pro Gly Leu His Gly Glu Phe Gly

50 55 60

Leu Pro Gly Pro Ala Gly Pro Arg Gly Glu Arg Gly Pro Pro Gly Glu

65 70 75 80

Ser Gly Ala Ala Gly Pro Thr Gly Pro Ile Gly Ser Arg Gly Pro Ser

85 90 95

Gly Pro Pro Gly Pro Asp Gly Asn Lys Gly Glu Pro Gly Val Val Gly

100 105 110

Ala Val Pro Gly Pro Ala Gly Lys Glu Gly Gly Lys Gly Pro Arg Gly

115 120 125

Glu Thr Gly Pro Ala Gly Arg Pro Gly Glu Val Gly Pro Pro Gly Pro

130 135 140

Pro Gly Pro Ala Gly Glu Lys Gly Ser Pro Gly Ala Asp Gly Pro Ala

145 150 155 160

Gly Ala Pro Gly Thr Pro Gly Pro Gln Gly Ile Ala Gly Gln Arg Gly

165 170 175

Val Val Gly Leu Pro Pro Gly Pro Ala Gly Lys Glu Gly Leu Arg Gly

180 185 190

Pro Arg Gly Asp Gln Gly Pro Val Gly Arg Thr Gly Glu Val Gly Ala

195 200 205

Val Gly Pro Pro Gly Phe Ala Gly Glu Lys Gly Pro Ser Gly Glu Ala

210 215 220

Gly Thr Ala Gly Pro Pro Gly Thr Pro Gly Pro Gln Gly Leu Leu Gly

225 230 235 240

Ala Pro Gly Ile Leu Gly Leu Pro Gly Leu Pro Gly Gln Arg Gly Glu

245 250 255

Arg Gly Phe Pro Gly Leu Pro Gly Pro Ser Gly Glu Pro Gly Lys Gln

260 265 270

Gly Pro Ser Gly Ala Ser Gly Glu Arg Gly Pro Pro Gly Pro Met Gly

275 280 285

Pro Pro Gly Leu Ala Gly Pro Pro Gly Glu Ser Gly Arg Glu Gly Ala

290 295 300

Pro Gly Ala Glu Gly Ser Pro Gly Arg Asp Gly Ser Pro Gly Ala Lys

305 310 315 320

Gly Asp Arg Gly Glu Thr Gly Pro Ala Gly Pro Pro Gly Ala Pro Gly

325 330 335

Ala Pro Gly Ala Pro Gly Pro Val Gly Pro Ala Gly Lys Ser Gly Asp

340 345 350

Arg Gly Glu Thr Gly Pro Ala Gly Pro Thr Gly Pro Val Gly Pro Val

355 360 365

Gly Ala Arg Gly Pro Ala Gly Pro Gln Gly Pro Arg Gly Asp Lys Gly

370 375 380

Glu Thr Gly Glu Gln Gly Asp Arg Gly Ile Lys Gly His Arg Gly Phe

385 390 395 400

Ser Gly Leu Gln Gly Pro Pro Gly Pro Pro Gly Ser Pro Gly Glu Gln

405 410 415

Gly Pro Ser Gly Ala Ser Gly Pro Ala Gly Pro Arg Gly Pro Pro Gly

420 425 430

Ser Ala Gly Ala Pro Gly Lys Asp Gly Leu Asn Gly Leu Pro Gly Pro

435 440 445

Ile Gly Pro Pro Gly Pro Arg Gly Arg Thr Gly Asp Ala Gly Pro Val

450 455 460

Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly

465 470 475 480

Glu Arg Gly Asp Leu Gly Pro Gln Gly Ile Ala Gly Gln Arg Gly Val

485 490 495

Val Gly Glu Arg Gly Glu Arg Gly Glu Arg Gly Ala Ser Gly Glu Arg

500 505 510

Gly Asp Leu Gly Pro Gln Gly Ile Ala Gly Gln Arg Gly Val Val Gly

515 520 525

Glu Arg Gly Glu Arg Gly Glu Arg Gly Ala Ser Gly Glu Arg Gly Asp

530 535 540

Leu Gly Pro Gln Gly Ile Ala Gly Gln Arg Gly Val Val Gly Glu Arg

545 550 555 560

Gly Glu Arg Gly Glu Arg Gly Ala Ser Gly Pro Pro Gly Pro Cys Cys

565 570 575

Gly Gly Gly Leu Glu His His His His His His

580 585

<210> 7

<211> 491

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 7

Asp Ala Pro Glu Glu Glu Asp His Val Leu Val Leu Arg Lys Ser Asn

1 5 10 15

Phe Ala Glu Ala Leu Ala Ala His Lys Tyr Leu Leu Val Glu Phe Tyr

20 25 30

Ala Pro Trp Cys Gly His Cys Lys Ala Leu Ala Pro Glu Tyr Ala Lys

35 40 45

Ala Ala Gly Lys Leu Lys Ala Glu Gly Ser Glu Ile Arg Leu Ala Lys

50 55 60

Val Asp Ala Thr Glu Glu Ser Asp Leu Ala Gln Gln Tyr Gly Val Arg

65 70 75 80

Gly Tyr Pro Thr Ile Lys Phe Phe Arg Asn Gly Asp Thr Ala Ser Pro

85 90 95

Lys Glu Tyr Thr Ala Gly Arg Glu Ala Asp Asp Ile Val Asn Trp Leu

100 105 110

Lys Lys Arg Thr Gly Pro Ala Ala Thr Thr Leu Pro Asp Gly Ala Ala

115 120 125

Ala Glu Ser Leu Val Glu Ser Ser Glu Val Ala Val Ile Gly Phe Phe

130 135 140

Lys Asp Val Glu Ser Asp Ser Ala Lys Gln Phe Leu Gln Ala Ala Glu

145 150 155 160

Ala Ile Asp Asp Ile Pro Phe Gly Ile Thr Ser Asn Ser Asp Val Phe

165 170 175

Ser Lys Tyr Gln Leu Asp Lys Asp Gly Val Val Leu Phe Lys Lys Phe

180 185 190

Asp Glu Gly Arg Asn Asn Phe Glu Gly Glu Val Thr Lys Glu Asn Leu

195 200 205

Leu Asp Phe Ile Lys His Asn Gln Leu Pro Leu Val Ile Glu Phe Thr

210 215 220

Glu Gln Thr Ala Pro Lys Ile Phe Gly Gly Glu Ile Lys Thr His Ile

225 230 235 240

Leu Leu Phe Leu Pro Lys Ser Val Ser Asp Tyr Asp Gly Lys Leu Ser

245 250 255

Asn Phe Lys Thr Ala Ala Glu Ser Phe Lys Gly Lys Ile Leu Phe Ile

260 265 270

Phe Ile Asp Ser Asp His Thr Asp Asn Gln Arg Ile Leu Glu Phe Phe

275 280 285

Gly Leu Lys Lys Glu Glu Cys Pro Ala Val Arg Leu Ile Thr Leu Glu

290 295 300

Glu Glu Met Thr Lys Tyr Lys Pro Glu Ser Glu Glu Leu Thr Ala Glu

305 310 315 320

Arg Ile Thr Glu Phe Cys His Arg Phe Leu Glu Gly Lys Ile Lys Pro

325 330 335

His Leu Met Ser Gln Glu Leu Pro Glu Asp Trp Asp Lys Gln Pro Val

340 345 350

Lys Val Leu Val Gly Lys Asn Phe Glu Asp Val Ala Phe Asp Glu Lys

355 360 365

Lys Asn Val Phe Val Glu Phe Tyr Ala Pro Trp Cys Gly His Cys Lys

370 375 380

Gln Leu Ala Pro Ile Trp Asp Lys Leu Gly Glu Thr Tyr Lys Asp His

385 390 395 400

Glu Asn Ile Val Ile Ala Lys Met Asp Ser Thr Ala Asn Glu Val Glu

405 410 415

Ala Val Lys Val His Ser Phe Pro Thr Leu Lys Phe Phe Pro Ala Ser

420 425 430

Ala Asp Arg Thr Val Ile Asp Tyr Asn Gly Glu Arg Thr Leu Asp Gly

435 440 445

Phe Lys Lys Phe Leu Glu Ser Gly Gly Gln Asp Gly Ala Gly Asp Asp

450 455 460

Asp Asp Leu Glu Asp Leu Glu Glu Ala Glu Glu Pro Asp Met Glu Glu

465 470 475 480

Asp Asp Asp Gln Lys Ala Val Lys Asp Glu Leu

485 490

<210> 8

<211> 895

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 8

Met Ile Ser Arg Thr Tyr Val Ile Asn Leu Ala Arg Arg Pro Asp Lys

1 5 10 15

Lys Asp Arg Ile Leu Ala Glu Phe Leu Lys Leu Lys Glu Lys Gly Val

20 25 30

Glu Leu Asn Cys Val Ile Phe Glu Ala Val Asp Gly Asn Asn Pro Glu

35 40 45

His Leu Ser Arg Phe Asn Phe Lys Ile Pro Asn Trp Thr Asp Leu Asn

50 55 60

Ser Gly Lys Pro Met Thr Asn Gly Glu Val Gly Cys Ala Leu Ser His

65 70 75 80

Trp Ser Val Trp Lys Asp Val Val Asp Cys Val Glu Asn Gly Thr Leu

85 90 95

Asp Lys Asp Cys Arg Ile Leu Val Leu Glu Asp Asp Val Val Phe Leu

100 105 110

Asp Asn Phe Met Glu Arg Tyr Gln Thr Tyr Thr Ser Glu Ile Thr Tyr

115 120 125

Asn Cys Asp Leu Leu Tyr Leu His Arg Lys Pro Leu Asn Pro Tyr Thr

130 135 140

Glu Thr Lys Ile Ser Thr His Ile Val Lys Pro Asn Lys Ser Tyr Trp

145 150 155 160

Ala Cys Ala Tyr Val Ile Thr Tyr Gln Cys Ala Lys Lys Phe Met Asn

165 170 175

Ala Asn Tyr Leu Glu Asn Leu Ile Pro Ser Asp Glu Phe Ile Pro Ile

180 185 190

Met His Gly Cys Asn Val Tyr Gly Phe Glu Lys Leu Phe Ser Asn Cys

195 200 205

Glu Lys Ile Asp Cys Tyr Ala Val Gln Pro Ser Leu Val Lys Leu Thr

210 215 220

Ser Asn Ala Phe Asn Asp Ser Glu Thr Phe His Ser Gly Ser Tyr Val

225 230 235 240

Pro Ser Asn Lys Phe Asn Phe Asp Thr Asp Lys Gln Phe Arg Ile Val

245 250 255

Tyr Ile Gly Pro Thr Lys Gly Asn Ser Phe His Arg Phe Thr Glu Tyr

260 265 270

Cys Lys Leu Tyr Leu Leu Pro Tyr Lys Val Ile Asp Glu Lys Glu Thr

275 280 285

Asn Asp Phe Val Ser Leu Arg Ser Glu Leu Gln Ser Leu Ser Glu Gln

290 295 300

Asp Leu Asn Thr Thr Leu Met Leu Val Val Ser Val Asn His Asn Asp

305 310 315 320

Phe Cys Asn Thr Ile Pro Cys Ala Pro Thr Asn Glu Phe Ile Asp Lys

325 330 335

Tyr Lys Gln Leu Thr Thr Asp Thr Asn Ser Ile Val Ser Ala Val Gln

340 345 350

Asn Gly Thr Asn Lys Thr Met Phe Ile Gly Trp Ala Asn Lys Ile Ser

355 360 365

Glu Phe Ile Asn His Tyr His Gln Lys Leu Thr Glu Ser Asn Ala Glu

370 375 380

Thr Asp Ile Asn Leu Ala Asn Leu Leu Leu Ile Ser Ser Ile Ser Ser

385 390 395 400

Asp Phe Asn Cys Val Val Glu Asp Val Glu Gly Asn Leu Phe Gln Leu

405 410 415

Ile Asn Glu Glu Ser Asp Ile Val Phe Ser Thr Thr Thr Ser Arg Val

420 425 430

Asn Asn Lys Leu Gly Lys Thr Pro Ser Val Leu Tyr Ala Asn Ser Asp

435 440 445

Ser Ser Val Ile Val Leu Asn Lys Val Glu Asn Tyr Thr Gly Tyr Gly

450 455 460

Trp Asn Glu Tyr Tyr Gly Tyr His Val Tyr Pro Val Lys Phe Asp Val

465 470 475 480

Leu Pro Lys Ile Tyr Leu Ser Ile Arg Ile Val Lys Asn Ala Asn Val

485 490 495

Thr Lys Ile Ala Glu Thr Leu Asp Tyr Pro Lys Glu Leu Ile Thr Val

500 505 510

Ser Ile Ser Arg Ser Glu His Asp Ser Phe Tyr Gln Ala Asp Ile Gln

515 520 525

Lys Phe Leu Leu Ser Gly Ala Asp Tyr Tyr Phe Tyr Ile Ser Gly Asp

530 535 540

Cys Ile Ile Thr Arg Pro Thr Ile Leu Lys Glu Leu Leu Glu Leu Asn

545 550 555 560

Lys Asp Phe Val Gly Pro Leu Met Arg Lys Gly Thr Glu Ser Trp Thr

565 570 575

Asn Tyr Trp Gly Asp Ile Asp Pro Ser Asn Gly Tyr Tyr Lys Arg Ser

580 585 590

Phe Asp Tyr Phe Asp Ile Ile Gly Arg Asp Arg Val Gly Cys Trp Asn

595 600 605

Val Pro Tyr Leu Ala Ser Val Tyr Leu Ile Lys Lys Ser Val Ile Glu

610 615 620

Gln Val Pro Asn Leu Phe Thr Glu Asn Ser His Met Trp Asn Gly Ser

625 630 635 640

Asn Ile Asp Met Arg Leu Cys His Asn Leu Arg Lys Asn Asn Val Phe

645 650 655

Met Tyr Leu Ser Asn Leu Arg Pro Tyr Gly His Ile Asp Asp Ser Ile

660 665 670

Asn Leu Glu Val Leu Ser Gly Val Pro Thr Glu Val Thr Leu Tyr Asp

675 680 685

Leu Pro Thr Arg Lys Glu Glu Trp Glu Lys Lys Tyr Leu His Pro Glu

690 695 700

Phe Leu Ser His Leu Gln Asn Phe Lys Asp Phe Asp Tyr Thr Glu Ile

705 710 715 720

Cys Asn Asp Val Tyr Ser Phe Pro Leu Phe Thr Pro Ala Phe Cys Lys

725 730 735

Glu Val Ile Glu Val Met Asp Lys Ala Asn Leu Trp Ser Lys Gly Gly

740 745 750

Asp Ser Tyr Phe Asp Pro Arg Ile Gly Gly Val Glu Ser Tyr Pro Thr

755 760 765

Gln Asp Thr Gln Leu Tyr Glu Val Gly Leu Asp Lys Gln Trp His Tyr

770 775 780

Val Val Phe Asn Tyr Val Ala Pro Phe Val Arg His Leu Tyr Asn Asn

785 790 795 800

Tyr Lys Thr Lys Asp Ile Asn Leu Ala Phe Val Val Lys Tyr Asp Met

805 810 815

Glu Arg Gln Ser Glu Leu Ala Pro His His Asp Ser Ser Thr Tyr Thr

820 825 830

Leu Asn Ile Ala Leu Asn Glu Tyr Gly Lys Glu Tyr Thr Ala Gly Gly

835 840 845

Cys Glu Phe Ile Arg His Lys Phe Ile Trp Gln Gly Gln Lys Val Gly

850 855 860

Tyr Ala Thr Ile His Ala Gly Lys Leu Leu Ala Tyr His Arg Ala Leu

865 870 875 880

Pro Ile Thr Ser Gly Lys Arg Tyr Ile Leu Val Ser Phe Val Asn

885 890 895

<210> 9

<211> 26

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

ccctcgagaa aagaccacca ttcttg 26

<210> 10

<211> 28

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

taagcggccg cttattagtg gtggtggt 28

<210> 11

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

cccaagcttg aaaaaaatgc accc 24

<210> 12

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

taagcggccg cttattattc caat 24

<210> 13

<211> 23

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

cccaagcttg aaaaaaatgg acg 23

<210> 14

<211> 23

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 14

taagcggccg cttattacaa ttc 23

<210> 15

<211> 27

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 15

cccaagcttg aaaaaaatga tctctag 27

<210> 16

<211> 28

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 16

taagcggccg cttattagtt aacgaaag 28

<210> 17

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 17

ggtgttccag gtgacttggg 20

<210> 18

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 18

ttagtggtgg tgatggtggt gttc 24

<210> 19

<211> 28

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 19

atggacgctc cagaagaaga agaccacg 28

<210> 20

<211> 30

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 20

ctagtagaac ccttcaacaa catcaagaat 30

<210> 21

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 21

atggacgctc cagaagaaga ag 22

<210> 22

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 22

ttacaattcg tccttaacag cc 22

<210> 23

<211> 27

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 23

atgatctcta gaacttacgt tatcaac 27

<210> 24

<211> 28

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 24

tagttaacga aagaaaccaa gatgtatc 28

Claims

1. Kluyveromyces lactis (Kluyveromyces lactis) expressing recombinant human-like collagen containing hydroxyproline and hydroxylysine, and is characterized in that: the food-grade expression host Kluyveromyces is taken as a chassis cell, and secretion expression of the hydroxylation recombinant human-like collagen is realized by co-transforming expression vectors containing proline hydroxylase alpha subunit, protein disulfide isomerase, lysine hydroxylase L230 and the recombinant human-like collagen; the hydroxylation recombinant human-like collagen is recombinant human-like collagen containing hydroxyproline and hydroxylysine; wherein the proline hydroxylase alpha subunit, the protease disulfide isomerase and the lysine hydroxylase are expressed in an intracellular constitutive mode, and the recombinant human-like collagen is expressed in a secretory mode; the preservation number of the kluyveromyces lactis expressing recombinant human-like collagen containing hydroxyproline and hydroxylysine is as follows: GDMCC NO 60895.

2. The production induction method of kluyveromyces lactis expressing recombinant human-like collagen containing hydroxyproline and hydroxylysine according to claim 1, characterized by comprising the steps of:

1) Inoculating: selecting single colony of the constructed Kluyveromyces lactis in YPD culture medium, and culturing at 28-30deg.C and 100-300rpm for 16-30 hr until OD600 reaches 20-30;

3) And (3) growing thalli: after the glucose is exhausted, glucose is added;

5) Centrifuging, and collecting supernatant of the fermentation broth to obtain recombinant human-like collagen fermentation broth containing hydroxyproline and hydroxylysine.

3. The production induction method of kluyveromyces lactis expressing recombinant human-like collagen containing hydroxyproline and hydroxylysine according to claim 2, wherein the production induction method comprises the following steps of: step 3) glucose is added in an amount of 0.1% -12%.

4. The production induction method of kluyveromyces lactis expressing recombinant human-like collagen containing hydroxyproline and hydroxylysine according to claim 2, wherein the production induction method comprises the following steps of: step 4) adding 0.1% -10% of galactose of plant origin.

5. The production induction method of kluyveromyces lactis expressing recombinant human-like collagen containing hydroxyproline and hydroxylysine according to claim 2, wherein the production induction method comprises the following steps of: the centrifugation in the step 5) is carried out for 2-30min at 3000g-10000 g.

6. Use of recombinant human-like collagen comprising hydroxyproline and hydroxylysine according to claim 1 for the preparation of a medicament for promoting cell adhesion and wound healing.