CN102443057A - Recombinant humanized collagen and its preparation method - Google Patents
Recombinant humanized collagen and its preparation method Download PDFInfo
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Abstract
The invention discloses a recombinant humanized collagen. Its amino acid sequence is as shown in SEQNO.3 and has 599 amino acids. The molecular weight is 55.0kDa. A preparation method of the recombinant humanized collagen comprises the following steps of: constructing a gene engineering strain for the expression of the recombinant humanized collagen to obtain a Pichia pastoris engineering strain; carrying out fermentation culture on the engineering strain, and realizing inducible expression of the recombinant humanized collagen to obtain a recombinant humanized collagen fermentation broth; and purifying the fermentation broth to obtain the recombinant humanized collagen. The humanized collagen gene Ge1 designed in the invention is a brand-new sequence which is greatly shorter than a natural humanized collagen gene (Kbp) in length. By the preparation method, operation simplification at the molecular level is realized, and the gene engineering strain fermentation production of the collagen is more easily realized. The humanized collagen gene Ge1 contains characteristics of a natural humanized collagen gene. And simultaneously, its expressed protein has excellent characteristics of a natural humanized collagen.
Description
Technical field
The invention belongs to biological technical field, particularly a kind of high-hydrophilic recombination human source collagen protein and preparation method thereof.
Background technology
Collagen protein (also claiming collagen) is the abundantest class protein family of content in the animal body.Collagen protein has very strong biological activity and biological function, can participate in migration, differentiation and the breeding of cell, makes reticular tissue have physical strength, and collagen protein can also promote the cell growth simultaneously, has hemostasis, biocompatibility and biodegradability.Based on these characteristics, collagen protein has a wide range of applications at pharmaceutical sanitary fields such as burn, wound, cornea disease, health care, beauty treatment, orthopedic, hard tissue repair, surface of a wound hemostasis, drug delivery, slow release methods.In addition, collagen protein also can be used as foodstuff additive, fodder additives, flocculation agent, photographic emulsion material, tensio-active agent and Carrier Materials of Immobilized Enzyme etc. and is applied to each industry and field, laboratory.
At present, the production of collagen protein mainly is to utilize acid, alkaline process to handle the tissue (pigskin, ox-hide, donkey hide, fish-skin etc.) of animal-origin, therefrom extracts collagen protein.But resulting product composition is complicated, is mainly used in feed and adds and fermentation culture medium for microbe.Mingrang Biological Science & Technology Co., Ltd., Sichuan Prov. adopts the directed shearing technique of enzyme, and the collagen protein of production is formed stable, biologically active.But the collagen protein product that above these methods obtain all has certain viral hidden danger (like mad cow disease, foot and mouth disease, swine fever epidemic disease, bird flu etc.); Be prone to cause the rejection of allosome xenogeneic when particularly being applied to human body, thereby limited the application of collagen protein aspect medical.
Along with the DNA recombinant technology develops rapidly, in order to make full use of the good characteristic of collagen protein, researcher begins to seek the collagen protein source beyond the animal itself.There are many scholars to utilize genetic engineering technique. select various host cells (comprising: Mammals, insect, yeast, intestinal bacteria, transgene tobacco, transgenic mouse, transgenic silkworm cell etc.) for use; Produce recombinant human collagen albumen, product has advantages such as security is good, favorable reproducibility, steady quality.But, exist many deficiencies and difficulty equally.For example the Tomita of the Hiroshima University of Japan utilizes the silk glands secretion of transgenic silkworm to produce human collagen, and the human collagen length of production only has 1/5 of real human collagen total length, and content has only about 1%.On the whole, expression amount is not high, the production cycle long and the cultivation difficulty is big, the cost height is ubiquitous problem when being the host with high animal and plant cells, and can not satisfies industrialization, requirement of mass production.Domestic have report (a kind of Human-like Collagen and a working method thereof of utilizing intestinal bacteria to produce Human-like Collagen; Contriver: Fan Daidi; Publication number: CN 1371919A, open day: on October 2nd, 2002, State Intellectual Property Office of the People's Republic of China).And bacterial expression system ubiquity generation pyrogeneous substance makes expression product be difficult to be applied to clinical; Albumen is often with the inclusion body formal representation; Cause the product purification difficulty, and the translation post-treatment of prokaryotic expression system is modified the system imperfection, the low deficiency that waits of the biological activity of expression product.
Summary of the invention
The object of the present invention is to provide a kind of recombination human source collagen protein that utilizes pichia pastoris to express, it has unique chemical structure and the performance that is superior to the animal body collagen protein, and the preparation method of this recombination human source collagen protein is provided simultaneously.
The technical solution that realizes the object of the invention is: a kind of recombination human source collagen protein, and aminoacid sequence such as SEQ NO.3, it contains 599 amino acid, and molecular weight is 55.0kDa.
A kind of preparation method of recombination human source collagen protein, step is following:
(1) structure of express recombinant people derived collagen protein gene engineering bacteria obtains the pichia pastoris genetic engineering bacterium;
(2) genetic engineering bacterium is carried out fermentation culture, realize the abduction delivering of recombination human source collagen protein, obtain containing the fermented liquid of recombination human source collagen protein;
(3) purification of recombinant human derived collagen albumen from fermented liquid.
The present invention compared with prior art; Its remarkable advantage: people's derived collagen protein gene Ge1 of (1) design is brand-new sequence; And be less than natural human collagen gene (number Kbp) on the length greatly; Realized simple to operateization, more be prone to realize the genetic engineering bacterium fermentative prodn of collagen protein at molecular level; (2) this people's derived collagen protein gene Ge1 comprises the characteristic of natural human collagen gene simultaneously, and its expressed proteins will have the excellent characteristic of natural human collagen protein; (3) aspect the proteic molecular length of people's derived collagen; Through preparing different repeat number series aiding connection gene recombination plasmids, can in further yeast expression research, realize expression production near the recombination human source collagen protein of the natural human collagen protein of bigger molecular weight; (4) preparation method of the different repeat number series aiding connection of this kind gene recombination plasmid; The protokaryon of only cutting ligation through simple vitro enzyme and having a mature technology transforms and screening, has realized being connected in series of goal gene (recombination human source collagen gene Ge1) of any repeat number; (5) the more existing many Using P CR technology of this method realizes that repeating to have more feasibility succeeds with Geng Yi. avoided reaching repeat to connect in the PCR operation of goal gene need highly difficult design of primers and condition exploration work; (6) but the Pichia yeast engineering of abduction delivering can realize the recombination human source collagen protein in born of the same parents with born of the same parents outside high level expression; Its cultivation is easy to amplify; And high density fermentation mode high yield target protein capable of using, secreting, expressing helps the separation and purification of expression product, reduces production costs; And it has certain posttranslational modification function to expressed foreign protein, like glycosylation etc.
Below in conjunction with accompanying drawing the present invention is described in further detail.
Description of drawings
Fig. 1 is that the SDS-PAGE of recombination human source collagen protein detects.
Fig. 2 is the gel permeation chromatography collection of illustrative plates in the purge process.
Fig. 3 is that the gel permeation chromatography collection of illustrative plates is used in the purification effect checking.
Fig. 4 is the mass spectroscopy of recombination human source collagen protein.
Fig. 5 is the UV spectrum of recombination human source collagen protein.
Fig. 6 is the ir spectra of recombination human source collagen protein.
Fig. 7 is the ESEM of recombination human source collagen protein.
Fig. 8 is that (X-coordinate concentration is relative concentration to the short NIH3T3 cell growth tendency of recombination human source collagen protein sponge.）。
Embodiment
Recombination human source collagen protein of the present invention, aminoacid sequence such as SEQ NO.3, it contains 599 amino acid, and molecular weight is 55.0kDa.
The preparation method of above-mentioned recombination human source collagen protein, step is following:
(1) structure of express recombinant people derived collagen protein gene engineering bacteria obtains the pichia pastoris genetic engineering bacterium, and the structure of its pichia pastoris genetic engineering bacterium is following:
1) based on human III type collagen protein α 1 chain collagen domain Gly-X-Y (the tripeptides Tumor-necrosis factor glycoproteins characteristic of glycocoll-X-Y), design and one section people's derived collagen of synthetic protein gene monomer Gel, its nucleotide sequence such as SEQNO.1; Cut connection through vitro enzyme then, utilize intestinal bacteria as cloning host, make up and contain the monomeric expression vector of six series connection people's derived collagen protein genes in the same way, this is six series connection people's monomeric nucleotide sequence of derived collagen protein gene such as SEQ NO.2 in the same way;
2) change recombinant expression vector over to expression that pichia pastoris carries out the recombination human source collagen protein; In the common micro-organisms center preservation of China Committee for Culture Collection of Microorganisms of depositary institution, deposit number is the pichia pastoris genetic engineering bacterium that obtains: CGMCC NO.5021.Classification called after pichia pastoris phaff Pichia Pastoris, preservation date is on June 29th, 2011, the address of depositary institution: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, postcode: 100101.
The structure detailed process of above-mentioned pichia pastoris genetic engineering bacterium is following.
1. design recombination human source collagen gene monomer (Ge1)
Its base sequence such as Fig. 1 (SEQ NO.1).Its length nucleic acid is 322bp, called after Ge1.This monomer is cloned in the SmaI site of plasmid pUC57, the recombinant plasmid called after pUC57Gel that obtains.The a pair of isocaudarner of selecting does
DraIII with
Van91I is positioned at the two ends of recombination human source collagen gene monomer (Ge1).Designed joint Al and A2, wherein the restriction enzyme site that contains of Al does
EcoRI with
DraThe restriction enzyme site that III, A2 contain does
NotI with
Van91I.The expression plasmid of selecting is pPIC9K.
2. will cut the goal gene monomer that obtains with a pair of isocaudarner enzyme and carry out external connection
Recombinant plasmid pUC57Gel is adopted CaCl
2Method transformed into escherichia coli DH5 α passes through the recombination bacillus coli that the LB plate screening that contains Amp obtains containing plasmid pUC57Gel, called after DH5 α/pUC57Gel.Liquid LB substratum incubated overnight DH5 α/pUC57Gel.Extract plasmid, obtain a large amount of pUC57Gel.With a pair of isocaudarner
DraIII with
Van9lI double digestion plasmid pUC57Gel; Electrophoresis reclaims the small segment of about 300bp, obtains a large amount of people's derived collagen protein gene monomers, and this part people's derived collagen protein gene monomer is connected with the T4DNA ligase enzyme; 4 ℃ are spent the night, and obtain containing the monomeric linker of different repeat number collagen genes.
3. utilize joint with the connection product that step 2 obtains, directly be connected into and contained the monomeric plasmid of goal gene. obtain the less recombinant plasmid of repeat number
Step 2 is obtained connecting product to join and contains pUC57Gel's
EcoRI/
DraIII double digestion rear electrophoresis reclaim the big fragment obtain with
EcoIn the linked system of the renaturation joint Al that the switchback of RI enzyme is received, 4 ℃ of connections of spending the night.Connect product and adopt CaCl
2Method transformed into escherichia coli Topl0F '; Through containing the LB plate screening positive colony of Amp; Through the plasmid that extracts the positive colony screen and carry out length that the restriction enzyme digestion and electrophoresis check contains plasmid and judge in the plasmid that recombination bacillus coli contained that obtains and comprise the monomeric repeat number of human-like collagen gene, the intestinal bacteria of the recombinant plasmid of 2,3,4 repeat numbers mainly appear including.To contain repeat number and be 3 plasmid and name and be pUC57A1G3, contain recombination bacillus coli called after the Top10F '/pUC57A1G3 of this pUC57A1G3 plasmid.
4. repeat number is less plasmid carries out the double connection of repeat number, obtains to contain the higher recombinant plasmid of repeat number of goal gene
Liquid LB substratum incubated overnight Top10F '/pUC57A1G3 extracts plasmid, gets pUC57A1G3 in a large number.With restriction enzyme
EcoRI with
Van9lI double digestion plasmid pUC57A1G3, electrophoresis reclaims the small segment of about 900bp, obtains a large amount of people's derived collagen protein gene triplet dna fragmentations.In addition with restriction enzyme
EcoRI with
DraIII double digestion plasmid pUC57A1G3, electrophoresis reclaims big fragment, obtains containing 3 and repeats the big fragment of people's monomeric A1G3 plasmid of derived collagen protein gene.Two kinds of segments that above-mentioned recovery is obtained connect with the T4DNA ligase enzyme, and 4 ℃ are spent the night, and the connection product that obtains directly adopts CaCl
2Method transformed into escherichia coli Top10F ' pulls screening and obtains containing the pUC57A1G6 recombination bacillus coli of (containing 6 people's derived collagen protein gene monomer multiple recombinant plasmids), called after Top10F '/pUC57A1G6 through the LB that contains Amp is flat.
The tandem gene of the suitable repeat number that 5. will build is cloned in the selected expression plasmid, the recombinant plasmid that acquisition can transformed host cell be used to express
Liquid LB substratum incubated overnight Top10F '/pUC57A1G6 extracts plasmid, obtains a large amount of DUC57A1G6.With restriction enzyme
EcoRI with
Van91I double digestion plasmid pUC57A1G6, electrophoresis reclaims the small segment of about 1800bp, obtains a large amount of people's derived collagen protein gene six conjuncted dna fragmentations.Join and contain pPIC9K's
EcoRI/
NotI double digestion rear electrophoresis reclaim the big fragment obtain with
NotIn the linked system of the renaturation joint A2 that the switchback of I enzyme is received, 4 ℃ of connections of spending the night.The connection product that obtains directly adopts CaCl
2Method transformed into escherichia coli Top10F '; Through the recombination bacillus coli that the LB plate screening that containing Amp obtains containing pPIC9KG6 (recombinant expression plasmid that contains 6 people's derived collagen protein gene monomers repetitions and joint Al and A2), called after Top10F '/pPIC9KG6.Again with liquid LB substratum incubated overnight T0p10F '/pPIC9KG6, extract plasmid. promptly can be used in a large number transforming pichia spp (
Pichia Pastoris) the pPIC9KG6 plasmid.
6. transform pichia pastoris
The expression vector that above-mentioned success makes up is used
SacI carries out linearizing to it, transforms after the GS115, on the MD flat board, obtains the fast type (Mut of methyl alcohol utilization
+) recombination yeast.Through bacterium colony PCR, can be tentatively definite, linearizing pPIC9KG6 has transformed into pichia spp GS115, and homologous recombination is gone into the karyomit(e) of GS115.
Electricity transforms the every MD flat board in back has tens single bacterium colonies, and it is dull and stereotyped to xerox the YPD that contains different concns G418, multiple copied recombination yeast 6 strains that to obtain anti-G418 concentration be 4. 00g/L.Select for use a wherein strain to express experiment.
7. SDS-PAGE detects
Subsequently reorganization zymic expression product is carried out the SDS-PAGE check and analysis; The result shows that the recombination human source collagen protein is with monomer (Fig. 1; Band 1, left arrow indication) and dimeric forms have (Fig. 1, band 1; The middle arrow indication) in fermented supernatant fluid, this characteristic with natural collagen protein is similar.
Recombination human source collagen protein of the present invention, the nucleotide sequence of its gene is seen SEQ NO.2, its aminoacid sequence is seen SEQ NO.3.The gene of this recombination human source collagen protein of encoding is to be the parent model with the human III type collagen protein; On the aminoacid sequence basis of its collagen domain; Is purpose to improve collagen protein water-soluble with improving expression amount, keeps the brand-new gene order of characteristic Design synthetic of collagen protein.Its pairing protein all is made up of Gly-X-Y tripeptides Tumor-necrosis factor glycoproteins; Various content of amino acids approach the natural human collagen protein; Change to some extent but put in order each other; And the hydrophobic nature amino acid except Pro (like Trp, Tyr, Phe, Leu, Ile, Val, Met etc.) is replaced by hydrophilic amino acid (like Asp, Asn, Glu, Gln etc.); To improve the wetting ability of recombination human source collagen protein, therefore have unique chemical structure and the performance that is superior to the animal body collagen protein.
(2) genetic engineering bacterium is carried out fermentation culture, realize the abduction delivering of recombination human source collagen protein, obtain containing the fermented liquid of recombination human source collagen protein;
The substratum of genetic engineering bacterium is:
1) seed culture medium (BMG): 100mM phosphoric acid buffer (pH6.0), 1.34%YNB, 4 * 10
-5The % vitamin H, 1% glycerine (V/V).
2) batch fermentation substratum (BSM): 85% H
3PO
426.7ml/L; CaSO
40.93g/L; K
2SO
418.2g/L; MgSO
47H
2O 14.9g/L; KOH 4.13g/L; Glycerine 40.0g/L adds PTM again after the sterilization
1Trace element.PTM wherein
1Trace element: CuSO
45H
2O 6.0g/L; NaI 0.08g/L; MnSO
4H
2O 3.0g/L; NaMoO
42H
2O 0.2g/L; H
3BO
30.02g/L; CoCl
20.5g/L; ZnCl
220.0g/L; FeSO
47H
2O 65.0g/L; Vitamin H 0.2g/L; H
2SO
45.0ml/L, with the membrane filtration degerming of 0.22 μ m, room temperature preservation.
3) feed supplement growth medium: 50% (W/V) glycerine, every liter of PTM that contains 12mL
1Trace element.
4) fermentation inducement substratum: 100% methyl alcohol, every liter of PTM that contains 12mL
1Trace element.
The fermentation culture conditions of genetic engineering bacterium and recombination human source collagen protein abduction delivering condition are:
Adopt in batches-the feed supplement cultural method; 28 ℃ of culture temperature; PH5.0; Promptly 1) BMG cultured strain (inoculum size 10%) is added to contain begin in the batch fermentation substratum fermentor tank to cultivate, regulating mixing speed is that 500 r/min-800 r/min, tank pressure are that 0.2-1.0bar, air flow quantity are 200 In/h-300 In/h, makes dissolved oxygen (DO)>30%.2) after carbon source exhausts, dissolved oxygen suddenly rises, and beginning flow feeding growth medium (the stream rate of acceleration is for keeping dissolved oxygen>20%) reaches 180 ~ 220g/L to the thalline weight in wet base, stops to add glycerine.3) glycerine exhausts the back and mends the fermentation inducement substratum and carry out abduction delivering, adds methyl alcohol speed and makes dissolved oxygen through regulating rotating speed, tank pressure, air flow quantity and stream>20%, induce the about 96-120h of fermentation after, finish fermentation, the collection fermented liquid.
The fermenting experiment result: the recombination human source collagen production is up to 16g/L.
The present invention utilizes the pichia pastoris genetic engineering bacterium to carry out the production of recombination human source collagen protein, has used pichia spp preference codon to guarantee the high expression level of recombination human source collagen protein; Adopt the fermentation condition and the methyl alcohol stream of above-mentioned optimization to add further raising output of strategy.
(3) purification of recombinant human derived collagen albumen from fermented liquid; The extraction purifying of recombination human source collagen protein is following: fermented liquid is carried out spinning, collect supernatant, supernatant is behind membrane filtration; With the separation and purification of Sephadex G100 gel filtration chromatography, obtain finished product through lyophilize again.The practical implementation process of the purifying of above-mentioned recombination human source collagen protein is:
1) fermented liquid is carried out centrifugal (8000 r/min) and separate, collect supernatant, supernatant is again through membrane filtration.
2) on the gel permeation chromatography post to supernatant through on appearance, elution step, collect elution peak (Fig. 2, the arrow indication contain the recombination human source collagen protein; SDS-PAGE detects and sees Fig. 1, band 3); The result shows that behind this purifying obtain recombination human source collagen protein dimer, apparent molecular weight is 120.0 kDa (Fig. 1, band 3, arrow indications), near its theoretical molecular (110.0 kDa).Its recovery reaches 93.6%, and purity reaches 99.5%.
3) for the above-mentioned conclusion of checking, the sample through the above-mentioned steps purifying is carried out filtration chromatography with Sephadex G100 gel once more, the result sees Fig. 3.Fig. 3 shows after the sample of above-mentioned steps purifying carries out gel permeation chromatography once more, still to have only a peak, and any specific conductivity peak do not occur, proves that both not had foreign protein in the sample does not have small-molecule substance or ion yet, and the effect of above-mentioned purification schemes obtains checking.
4) collect target protein through lyophilize.
The expressed foreign protein content of pichia pastoris genetic engineering bacterium of the present invention is few, is beneficial to the separation and purification of recombination human source collagen protein.
The water-soluble detection of recombination human source collagen protein of the present invention: the proteic water-soluble test experience of recombinant human derived collagen is shown; Under the room temperature; The saturation concentration of recombination human source collagen protein in water reaches 40.06 g/L, comes product-derived (7.63g/L) far above natural animal.
The structural characterization of recombination human source collagen protein of the present invention: the recombination human source collagen protein after the separation and purification is carried out amino acid analysis (amino acid analysis of recombination human source collagen protein is seen table 1), and the result shows that the detected value of recombination human source collagen, amino acid composition is consistent with its theoretical value.
The amino acid analysis of table 1 recombination human source collagen protein
Mass spectroscopy (Fig. 4) result shows that recombination human source collagen monomer molecular weight is 56460.056Da; The dimer molecule amount is 112920.339Da; The recombination human source collagen protein of design contains 599 amino acid in theory; Molecular weight is 55.0kDa, and actual accurate molecular weight (56460.056Da) is more bigger than theoretical value, is attributable to pichia spp secretory protein has been carried out the glycosylation modified of O-connection and N-connection.
Recombination human source collagen protein uv scan (Fig. 5) shows that itself and natural collagen protein have identical uv-absorbing.
The wave number of acid amides A from ir spectra (Fig. 6), acid amides B, acid amides I, acid amides II, acid amides III can judge that recombination human source collagen protein and natural collagen protein have similar constitutional features.
ESEM (Fig. 7) shows that the recombination human source collagen protein is spongy structure, indicates that it possesses the potential quality as biomedical material.
The promoter action of recombination human source collagen protein cell growth of the present invention: MTT detects (MTT test experience data are seen table 2): experimental result shows: certain density recombination human source collagen protein sponge and pigskin collagen protein sponge and NINH 3T3 cell have good consistency, can promote the growth of NIH3T3 cell; Below 10mg/mL concentration, concentration is from low to high the time, and (Fig. 8, Trendline a) promotes that with pigskin collagen protein sponge (Fig. 8, Trendline b) growth tendency of NIH3T3 cell is to raise gradually to the recombination human source collagen protein sponge.
Table 2 MTT test experience data
SEQ NO.1: recombination human source collagen gene monomer nucleotide sequence
< 110>Institutes Of Technology Of Nanjing
< 120>a kind of recombination human source collagen protein and working method thereof
<130> 2011
<160> 1
<170> PatentIn?version?3.3
<210> 1
<211> 322
<212> DNA
< 213>artificial sequence
<400> 1
agaggtccac?ccggtgagcc?aggtaaccca?ggttctccag?gaaaccaagg?tcaacctgga 60
aacaagggtt?ctcctggaaa?cccaggtcaa?cctggtaacg?agggacaacc?aggtcaacct 120
ggtcaaaacg?gtcaaccagg?tgagcctgga?tctaacggtc?ctcaaggttc?ccaaggtaac 180
ccaggtaaga?acggtcaacc?aggttctcca?ggttcccaag?gttctcctgg?aaaccaaggt 240
tctccaggtc?aaccaggtaa?cccaggtcaa?cctggagagc?aaggtaagcc?aggtaaccaa 300
ggtccagccg?gtggttaaag?aa 322
SEQ NO.2: recombination human source collagen gene nucleotide sequence
< 110>Institutes Of Technology Of Nanjing
< 120>a kind of recombination human source collagen protein and working method thereof
<130> 2011
<160> 1
<170> PatentIn?version?3.3
<210> 1
<211> 1797
<212> DNA
< 213>artificial sequence
<400> 1
ggtccacccg?gtgagccagg?taacccaggt?tctccaggaa?accaaggtca?acctggaaac 60
aagggttctc?ctggaaaccc?aggtcaacct?ggtaacgagg?gacaaccagg?tcaacctggt 120
caaaacggtc?aaccaggtga?gcctggatct?aacggtcctc?aaggttccca?aggtaaccca 180
ggtaagaacg?gtcaaccagg?ttctccaggt?tcccaaggtt?ctcctggaaa?ccaaggttct 240
ccaggtcaac?caggtaaccc?aggtcaacct?ggagagcaag?gtaagccagg?taaccaaggt 300
ccagccggtg?agccaggtaa?cccaggttct?ccaggaaacc?aaggtcaacc?tggaaacaag 360
ggttctcctg?gaaacccagg?tcaacctggt?aacgagggac?aaccaggtca?acctggtcaa 420
aacggtcaac?caggtgagcc?tggatctaac?ggtcctcaag?gttcccaagg?taacccaggt 480
aagaacggtc?aaccaggttc?tccaggttcc?caaggttctc?ctggaaacca?aggttctcca 540
ggtcaaccag?gtaacccagg?tcaacctgga?gagcaaggta?agccaggtaa?ccaaggtcca 600
gccggtgagc?caggtaaccc?aggttctcca?ggaaaccaag?gtcaacctgg?aaacaagggt 660
tctcctggaa?acccaggtca?acctggtaac?gagggacaac?caggtcaacc?tggtcaaaac 720
ggtcaaccag?gtgagcctgg?atctaacggt?cctcaaggtt?cccaaggtaa?cccaggtaag 780
aacggtcaac?caggttctcc?aggttcccaa?ggttctcctg?gaaaccaagg?ttctccaggt 840
caaccaggta?acccaggtca?acctggagag?caaggtaagc?caggtaacca?aggtccagcc 900
ggtgagccag?gtaacccagg?ttctccagga?aaccaaggtc?aacctggaaa?caagggttct 960
cctggaaacc?caggtcaacc?tggtaacgag?ggacaaccag?gtcaacctgg?tcaaaacggt 1020
caaccaggtg?agcctggatc?taacggtcct?caaggttccc?aaggtaaccc?aggtaagaac 1080
ggtcaaccag?gttctccagg?ttcccaaggt?tctcctggaa?accaaggttc?tccaggtcaa 1140
ccaggtaacc?caggtcaacc?tggagagcaa?ggtaagccag?gtaaccaagg?tccagccggt 1200
gagccaggta?acccaggttc?tccaggaaac?caaggtcaac?ctggaaacaa?gggttctcct 1260
ggaaacccag?gtcaacctgg?taacgaggga?caaccaggtc?aacctggtca?aaacggtcaa 1320
ccaggtgagc?ctggatctaa?cggtcctcaa?ggttcccaag?gtaacccagg?taagaacggt 1380
caaccaggtt?ctccaggttc?ccaaggttct?cctggaaacc?aaggttctcc?aggtcaacca 1440
ggtaacccag?gtcaacctgg?agagcaaggt?aagccaggta?accaaggtcc?agccggtgag 1500
ccaggtaacc?caggttctcc?aggaaaccaa?ggtcaacctg?gaaacaaggg?ttctcctgga 1560
aacccaggtc?aacctggtaa?cgagggacaa?ccaggtcaac?ctggtcaaaa?cggtcaacca 1620
ggtgagcctg?gatctaacgg?tcctcaaggt?tcccaaggta?acccaggtaa?gaacggtcaa 1680
ccaggttctc?caggttccca?aggttctcct?ggaaaccaag?gttctccagg?tcaaccaggt 1740
aacccaggtc?aacctggaga?gcaaggtaag?ccaggtaacc?aaggtccagc?cggtggt 1797
SEQ NO.3: recombination human source collagen, amino acid sequence
< 110>Institutes Of Technology Of Nanjing
< 120>a kind of recombination human source collagen protein and working method thereof
<130> 2011
<160> 1
<170> PatentIn?version?3.3
<210> 1
<211> 599
<212> PRT
< 213>artificial sequence
<400> 1
Gly?Pro?Pro?Gly?Glu?Pro?Gly?Asn?Pro?Gly?Ser?Pro?Gly?Asn?Gln?Gly
1 5 10 15
Gln?Pro?Gly?Asn?Lys?Gly?Ser?Pro?Gly?Asn?Pro?Gly?Gln?Pro?Gly?Asn
20 25 30
Glu?Gly?Gln?Pro?Gly?Gln?Pro?Gly?Gln?Asn?Gly?Gln?Pro?Gly?Glu?Pro
35 40 45
Gly?Ser?Asn?Gly?Pro?Gln?Gly?Ser?Gln?Gly?Asn?Pro?Gly?Lys?Asn?Gly
50 55 60
Gln?Pro?Gly?Ser?Pro?Gly?Ser?Gln?Gly?Ser?Pro?Gly?Asn?Gln?Gly?Ser
65 70 75 80
Pro?Gly?Gln?Pro?Gly?Asn?Pro?Gly?Gln?Pro?Gly?Glu?Gln?Gly?Lys?Pro
85 90 ?95
Gly?Asn?Gln?Gly?Pro?Ala?Gly?Glu?Pro?Gly?Asn?Pro?Gly?Ser?Pro?Gly
100 105 110
Asn?Gln?Gly?Gln?Pro?Gly?Asn?Lys?Gly?Ser?Pro?Gly?Asn?Pro?Gly?Gln
115 120 125
Pro?Gly?Asn?Glu?Gly?Gln?Pro?Gly?Gln?Pro?Gly?Gln?Asn?Gly?Gln?Pro
130 135 140
Gly?Glu?Pro?Gly?Ser?Asn?Gly?Pro?Gln?Gly?Ser?Gln?Gly?Asn?Pro?Gly
145 150 155 ?160
Lys?Asn?Gly?Gln?Pro?Gly?Ser?Pro?Gly?Ser?Gln?Gly?Ser?Pro?Gly?Asn
165 170 ?175
Gln?Gly?Ser?Pro?Gly?Gln?Pro?Gly?Asn?Pro?Gly?Gln?Pro?Gly?Glu?Gln
180 185 ?190
Gly?Lys?Pro?Gly?Asn?Gln?Gly?Pro?Ala?Gly?Glu?Pro?Gly?Asn?Pro?Gly
195 200 205
Ser?Pro?Gly?Asn?Gln?Gly?Gln?Pro?Gly?Asn?Lys?Gly?Ser?Pro?Gly?Asn
210 215 ?220
Pro?Gly?Gln?Pro?Gly?Asn?Glu?Gly?Gln?Pro?Gly?Gln?Pro?Gly?Gln?Asn
225 230 ?235 240
Gly?Gln?Pro?Gly?Glu?Pro?Gly?Ser?Asn?Gly?Pro?Gln?Gly?Ser?Gln?Gly
245 250 ?255
Asn?Pro?Gly?Lys?Asn?Gly?Gln?Pro?Gly?Ser?Pro?Gly?Ser?Gln?Gly?Ser
260 265 ?270
Pro?Gly?Asn?Gln?Gly?Ser?Pro?Gly?Gln?Pro?Gly?Asn?Pro?Gly?Gln?Pro
275 280 285
Gly?Glu?Gln?Gly?Lys?Pro?Gly?Asn?Gln?Gly?Pro?Ala?Gly?Glu?Pro?Gly
290 295 300
Asn?Pro?Gly?Ser?Pro?Gly?Asn?Gln?Gly?Gln?Pro?Gly?Asn?Lys?Gly?Ser
305 310 ?315 320
Pro?Gly?Asn?Pro?Gly?Gln?Pro?Gly?Asn?Glu?Gly?Gln?Pro?Gly?Gln?Pro
325 330 335
Gly?Gln?Asn?Gly?Gln?Pro?Gly?Glu?Pro?Gly?Ser?Asn?Gly?Pro?Gln?Gly
340 345 350
Ser?Gln?Gly?Asn?Pro?Gly?Lys?Asn?Gly?Gln?Pro?Gly?Ser?Pro?Gly?Ser
355 360 365
Gln?Gly?Ser?Pro?Gly?Asn?Gln?Gly?Ser?Pro?Gly?Gln?Pro?Gly?Asn?Pro
370 375 ?380
Gly?Gln?Pro?Gly?Glu?Gln?Gly?Lys?Pro?Gly?Asn?Gln?Gly?Pro?Ala?Gly
385 390 ?395 400
Glu?Pro?Gly?Asn?Pro?Gly?Ser?Pro?Gly?Asn?Gln?Gly?Gln?Pro?Gly?Asn
405 410 ?415
Lys?Gly?Ser?Pro?Gly?Asn?Pro?Gly?Gln?Pro?Gly?Asn?Glu?Gly?Gln?Pro
420 425 430
Gly?Gln?Pro?Gly?Gln?Asn?Gly?Gln?Pro?Gly?Glu?Pro?Gly?Ser?Asn?Gly
435 440 445
Pro?Gln?Gly?Ser?Gln?Gly?Asn?Pro?Gly?Lys?Asn?Gly?Gln?Pro?Gly?Ser
450 455 460
Pro?Gly?Ser?Gln?Gly?Ser?Pro?Gly?Asn?Gln?Gly?Ser?Pro?Gly?Gln?Pro
465 470 ?475 480
Gly?Asn?Pro?Gly?Gln?Pro?Gly?Glu?Gln?Gly?Lys?Pro?Gly?Asn?Gln?Gly
485 490 ?495
Pro?Ala?Gly?Glu?Pro?Gly?Asn?Pro?Gly?Ser?Pro?Gly?Asn?Gln?Gly?Gln
500 505 510
Pro?Gly?Asn?Lys?Gly?Ser?Pro?Gly?Asn?Pro?Gly?Gln?Pro?Gly?Asn?Glu
515 520 525
Gly?Gln?Pro?Gly?Gln?Pro?Gly?Gln?Asn?Gly?Gln?Pro?Gly?Glu?Pro?Gly
530 535 ?540
Ser?Asn?Gly?Pro?Gln?Gly?Ser?Gln?Gly?Asn?Pro?Gly?Lys?Asn?Gly?Gln
545 550 ?555 560
Pro?Gly?Ser?Pro?Gly?Ser?Gln?Gly?Ser?Pro?Gly?Asn?Gln?Gly?Ser?Pro
565 570 ?575
Gly?Gln?Pro?Gly?Asn?Pro?Gly?Gln?Pro?Gly?Glu?Gln?Gly?Lys?Pro?Gly
580 585 590
Asn?Gln?Gly?Pro?Ala?Gly?Gly
595
Claims (6)
1. a recombination human source collagen protein is characterized in that aminoacid sequence such as SEQ NO.3, and it contains 599 amino acid, and molecular weight is 55.0kDa.
2. the preparation method of a recombination human source collagen protein is characterized in that step is following:
(1) structure of express recombinant people derived collagen protein gene engineering bacteria obtains the pichia pastoris genetic engineering bacterium;
(2) genetic engineering bacterium is carried out fermentation culture, realize the abduction delivering of recombination human source collagen protein, obtain containing the fermented liquid of recombination human source collagen protein;
(3) purification of recombinant human derived collagen albumen from fermented liquid.
3. the preparation method of recombination human source collagen protein according to claim 2 is characterized in that the structure of said express recombinant people's derived collagen protein gene engineering bacteria is following:
1) based on the tripeptides Tumor-necrosis factor glycoproteins characteristic of human III type collagen protein α 1 chain collagen domain Gly-X-Y, design is one section people's derived collagen of synthetic protein gene monomer also, its nucleotide sequence such as SEQ NO.1; Cut connection through vitro enzyme then, utilize intestinal bacteria as cloning host, make up and contain the monomeric expression vector of six series connection people's derived collagen protein genes in the same way, this is six series connection people's monomeric nucleotide sequence of derived collagen protein gene such as SEQ NO.2 in the same way;
2) change recombinant expression vector over to expression that pichia pastoris carries out the recombination human source collagen protein; In the common micro-organisms center preservation of China Committee for Culture Collection of Microorganisms of depositary institution, deposit number is the pichia pastoris genetic engineering bacterium that obtains: CGMCC NO. 5021.
4. the preparation method of recombination human source collagen protein according to claim 2 is characterized in that the substratum of genetic engineering bacterium is:
1) seed culture medium: 100mM phosphoric acid buffer (pH6.0), 1.34%YNB, 4 * 10
-5The % vitamin H, 1% glycerine (V/V);
2) batch fermentation substratum: 85% H
3PO
426.7ml/L; CaSO
40.93g/L; K
2SO
418.2g/L; MgSO
47H
2O 14.9g/L; KOH 4.13g/L; Glycerine 40.0g/L adds PTM again after the sterilization
1Trace element; PTM wherein
1Trace element: CuSO
45H
2O 6.0g/L; NaI 0.08g/L; MnSO
4H
2O 3.0g/L; NaMoO
42H
2O 0.2g/L; H
3BO
30.02g/L; CoCl
20.5g/L; ZnCl
220.0g/L; FeSO
47H
2O 65.0g/L; Vitamin H 0.2g/L; H
2SO
45.0ml/L, with the membrane filtration degerming of 0.22 μ m, room temperature preservation;
3) feed supplement growth medium: 50% (W/V) glycerine, every liter of PTM that contains 12mL
1Trace element;
4) fermentation inducement substratum: 100% methyl alcohol, every liter of PTM that contains 12mL
1Trace element.
5. the preparation method of recombination human source collagen protein according to claim 2 is characterized in that the fermentation culture conditions of genetic engineering bacterium and recombination human source collagen protein abduction delivering condition are:
Adopt in batches-the feed supplement cultural method; 28 ℃ of culture temperature; PH5.0; Promptly 1) bacterial classification of seed culture medium being cultivated adds to contain by 10% inoculum size and begins in the batch fermentation substratum fermentor tank to cultivate, and regulating mixing speed is that 500 r/min-800 r/min, tank pressure are that 0.2-1.0bar, air flow quantity are 200 In/h-300 In/h, makes dissolved oxygen>30%; 2) after carbon source exhausts, dissolved oxygen suddenly rises, and beginning flow feeding growth medium, the stream rate of acceleration is for keeping dissolved oxygen>20%, reach 180 ~ 220g/L to the thalline weight in wet base, stop to add glycerine; 3) glycerine exhausts the back and mends the fermentation inducement substratum and carry out abduction delivering, adds methyl alcohol speed and makes dissolved oxygen through regulating rotating speed, tank pressure, air flow quantity and stream>20%, induce fermentation 96-120h after, finish fermentation, collect fermented liquid.
6. the preparation method of recombination human source collagen protein according to claim 2; The extraction purifying that it is characterized in that the recombination human source collagen protein is following: fermented liquid is carried out spinning; Collect supernatant; Supernatant with the separation and purification of Sephadex G100 gel filtration chromatography, obtains finished product through lyophilize again behind membrane filtration.
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Effective date of registration: 20150619 Address after: 214504, 2 south side of Textile Road, eastern suburbs village, Jingjiang City, Jiangsu, Taizhou Patentee after: JIANGSU JLAND BIOTECH CO., LTD. Address before: Pukou Lijing Road District of Nanjing City, Jiangsu province 210032 No. 2 R & D building A, 8 floor, 9 floor Patentee before: High & Tech Development Co., Ltd. Nanjing University of Science and Technology |