CN1196712C - Human like collagen and production method thereof - Google Patents

Human like collagen and production method thereof Download PDF

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CN1196712C
CN1196712C CN01106757.8A CN01106757A CN1196712C CN 1196712 C CN1196712 C CN 1196712C CN 01106757 A CN01106757 A CN 01106757A CN 1196712 C CN1196712 C CN 1196712C
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gap
gpp
gpa
gsa
human
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CN1371919A (en
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范代娣
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XI'AN GIANT BIOGENE TECHNOLOGY CO., LTD.
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范代娣
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]

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Abstract

The present invention discloses human like collagen protein, and a method for producing the human like collagen protein by genetic engineering bacteria. The method for producing the human like collagen protein comprises the following steps that the engineering bacteria of the human like collagen protein is constructed; the engineering bacteria are fermented and cultivated; target protein is obtained by the inducement, the expression and the refining of the human like collagen protein. The recombination protein obtained by the method has high expression in single cells, and the human like collagen protein is in a spiral structure and is different from a crossing structure of the original protein of an animal body so as to have fine workability without the molecular weight change; thereby, the human like collagen protein can be processed into a suture thread for an operation, artificial skin, a rubber sheet coating layer, an artificial organ coating layer, etc., and can be also combined with silver halide, dye, etc., to form coating materials with good surface viscosity.

Description

A kind of Human-like Collagen and production method thereof
The method that the present invention relates to a kind of Human-like Collagen and produce Human-like Collagen by genetic engineering bacterium.
Collagen protein is one of intrinsic albumen of human body, its content in skin is maximum, because collagen protein inherent bio-compatibility, biological degradability and absorptivity, short new cell form and short epithelial cell forms function, collagen protein is constantly widened in the potential use of medical field, beauty treatment, makeup, health products trade, but the animal body collagen protein is a kind of scleroprotein (water is insoluble), and with advancing age, internal double bonds is more and more, and is simultaneously also more and more harder.Generally, the collagen protein that obtains through enzymolysis is a water-insoluble protein, also can not change heavily dissolving under the molecular weight situation.Therefore, processability is very weak, has directly limited the exploitation of many potential uses.In addition, collagen protein from animal body can not be got rid of viral hidden danger, all the time the characteristic that has simultaneously bovine collagen, in order to reduce its immune rejection, scientists once idea is obtained collagen protein from people's placenta, still, this research is subjected to the resistance of world human rights organization and FDA, because can not definitely detect the virus and the bacterium of each placenta tissue.
In the prior art, the production of domestic and international market short run collagen protein is all from the extract of animal skin, heel string, cartilage and people's placenta.Its extraction process divided for three steps, one, the dissolving; Two, enzyme is handled; Three, purifying.May have various viruses but extract the class collagen protein, so the various products of animal skin and internal organs extract are directly used in the people and know from experience and cause serious hidden danger by animal skin.
External report is produced collagen protein research with recombinant technology to be had: recombinant yeast cell is produced accurate human collagen II, recombinant mammalian cells is produced accurate human collagen I, II, the III type, the reorganization cerevisiae is produced accurate human collagen VI, recombinant insect cell is produced accurate human collagen II, expression amount is 50mg/ml, human tumor cells is produced accurate people's fiber collagen 1A1 and 1A2, because animal cell culture cost costliness, the cycle is long, general 15 days even longer, the scale of cultivating can only be limited to experimental size in addition, satisfy industrialization, demand in enormous quantities almost is impossible fully.In addition, these recombination classes collagen proteins all must have the participation of back saccharase, must have one after conversion process, up to the present, with recombinant animal cells produce collagen protein must 8 kinds after the participation of saccharase, conversion process is quite complicated.
The object of the invention provides a kind of Human-like Collagen that can obtain high expression level in unicellular, and it has unique chemical structure and the performance that is better than the animal body collagen protein, and this glue class people is provided former proteic production method simultaneously.
For achieving the above object, the technical solution adopted in the present invention is:
A kind of human-like collagen; ：HDP VVL QRR DWE NPG VTQ LNR HLA HAH PPF ASD HPM GAPGPA GAP GPP GAP GPA GPP GSA GAP GPP GAP GPA GPP GSAGAP GPP GAP GPA GPP GSA GAP GPP GAP GPA GPP GSA GAPGPP GAP GPA GPP GSA GAP GPP GAP GPA GPP GSA GAP GPPGAP GPA GPP GSA GAP GPP GAP GPA GPP GSA GAP GPP GAPGPA GPP GSA GAP GPP GAP GPA GPP GSA GAP GPP GAP GPAGPP GSA GAP GPP GAP GPA GPP GSA GAP GPP GAP GPA GPPGSA GAP GPP GAP GPA GPP GSA GAP GPP GAH GPA GAL GAHGPA GPL GPA GPP GSA GAP GAH GPA GPL GAH GPA GPL GAHGPA GPL GAH GPA GPL GAP GPA GPP GSA GAP GPP GAP GPAGPP GSA GAP GPP GAP GPA GPP GSA GAP GPP GAP GPA GPPGSA GAP GPP GAP GPA GPP GSA GAP GPP GAP GPA GPP GSAGAP GPP GAP GPA GPP GSA GAP GPP GAP GPA GPP GSA GAPGPP GAP GPA GPP GSA GAP GPP GAP GPA GPP GSA GAP GPPGAH GPA GPL GAH GPA GPL GAH GPA GPL GAH GPA GPL GAPGPA GSA GAP GPP GAP GPA GPP GSA GAP GPP GAP GPA GPPGSA GAP GPP GAP GPA GPP GSA GAP GPP GAP GPA GPP GSAGAP GPP GAP GPA GPP GSA GAP GPP GAP GPA GPP GSA GAPGPP GAP GPA GPP GSA GAP GPP GAP GPA GPP GSA GAP GPPGAP GPA GPP GSA GAP GPP GAP GPA GPP GSA GAP GPP GAPGPA GPP GSA GAP GPP GAH GPA GPL GAH GPA GPL GAH GPAGPL GAH GPA GPL GAP GPA GPP GSA GAP GPP GAP GPA GPPGSA GAP GPP GAP GPA GPP GSA GAP GPP GAP GPA GPP GSAGAP GPP GAP GPA GPP GSA GAP GPP GSA GAP GPP GAP GPAGPP GSA GAP GPP GAP GPA GPP GSA GAP GPP GAP GPA GPPGSA GAP GPP GAP GPA GPP GSA GAP GPP GAP GPA GPP GSAGAP GPP GAH GPA GPL GAH GPA GPL GAH GPA GPL GAM GAPGAT GLS AGA THG LVT CGL
Its structure is three chains, triple-helix structure, and its length nucleic acid is 1071bp.
A kind of production method of Human-like Collagen, its special character is: it may further comprise the steps
1), the structure of the engineering bacteria of Human-like Collagen;
2), the fermentation culture of engineering bacteria;
3), Human-like Collagen inducing and expressing;
4), the purification of Human-like Collagen.
A kind of production method of Human-like Collagen, its special character is: the structure of the engineering bacteria of described Human-like Collagen is as follows:
1), a fragment gene of the known collagen protein sequence of human body extracted and modifies after, specific again repetition connects then, the DNA reorganization;
2), recombinant DNA is transferred to intestinal bacteria;
A kind of production method of Human-like Collagen, its special character is: the fermention medium of described engineering bacteria is:
Yeast powder 10-30g/L glucose 15-38g/L
NaHPO 4 2.5-8.7g/L (NH 4) 2SO 4 3.6-5.6g/L
FeCl 3 0.8-1.6g/L CoCl 2.6H 2O 0.1-0.3g/L
ZnSO 4 0.1-0.3g/L
A kind of production method of Human-like Collagen, its special character is; The fermentation culture conditions of described engineering bacteria is:
34 ℃ of culture temperature, PH7.0 dissolved oxygen DO 20%, air flow quantity is 1VVM-4VVM, mixing speed 450r/min-1000r/min, inoculum size 5%, seed culture medium, LB substratum, kantlex 0.5mg/L, glucose 5g/L, be advisable with glucose concn control 1.5%, be controlled to be 40% with the DO amount and be advisable, work as OD 600Value be 150 o'clock harvested cells.
A kind of production method of Human-like Collagen, its special character are that the purification of described Human-like Collagen is as follows:
Cell after the damping fluid extracting is used in high pressure cell homogenate then, after for the first time the formic acid damping fluid is saltoutd, then carries out ion-exchange through centrifugal collection, and refining and remove thermal source again through metal ion-chelant absorption, elaboration goes lyophilize.
Below in conjunction with specific embodiment, it is as follows to describe the present invention in detail:
The production method of Human-like Collagen comprises: the structure of the engineering bacteria of Human-like Collagen; The fermentation culture of engineering bacteria and inducing; The purification of collagen protein obtains target protein.
One, the structure of the engineering bacteria of Human-like Collagen
(1), prepare plasmid DNA from the E.coLi bacterial strain:
When preparing on a small scale: plasmid DNA prepares through acid-soluble method from the 1.5ml culture of Escherichia coli.
When mass preparation: the carrier bacterial strain is containing overnight incubation on the substratum of antibiotic, centrifugal collecting cell, centrifugal speed 10000r/min5 minute, again be suspended in ice-cold TE buffer soln (the 10ml EDTA of 10ml then, pH 8,) centrifugal again, resuspending is in 4ml TES (in TE and 20% the sucrose liquid), add lytic enzyme and cultivate adding 2ml0.5M EDTA after 5 minutes, cultivate after 10 minutes and to add protein resolvase and dissolving damping fluid, after cultivate, centrifugal collection (35000r/min collects 2hr) afterwards supernatant liquor change centrifuge tube over to and add CsCl etc. in proportion.
(2), DNA alcohol is analysed
The sodium-acetate (pH7.5) of adding 10% and the cold ethanol of 3 times of volumes in the DNA sample are cultivated 30min for-70 ℃, and this moment, DNA separated out.Behind 4 ℃ of centrifugal 5min supernatant liquor alcohol is again analysed by (cold ethanol), dissolve in again in the DNA damping fluid afterwards with 80%.
(3), DNA digestion with restriction enzyme
DNA is used enzymic digestion with restriction enzyme in its corresponding damping fluid, DNA concentration is kept 4%, 37 ℃ and is cultivated 3hr, damping fluid proportioning (0.37mol/L Tris, 0.74mol/L Potassium ethanoate), 0.2mol/L magnesium acetate 0.05mol/L DTT (dithiothreitol (DTT)), PH=8.0
(4), DNA electrophoretic analysis
In the DNA sample, add electrophoretic buffer, move 20hr under the 50V constant voltage.The damping fluid proportioning is with conventional electrophoretic buffer.
Embodiment 1:D1 collagen type
Digest with the synthetic PPD1121 plasmid of restriction enzyme HindIII, separate with agarose then, get the collagen gene fragment, sew up with postdigestive plasmid PSC101 behind the purifying, sew up DNA and change among e. coli bl21 or the HB101, receive mycin and carry out clonal selection with blocking.Clone strain is cultivated again, carried out resistance identification and protein expression analysis with SDS-Page.
(5), DNA connects
Under the effect of DNA stitching tackiness agent, under room temperature, react 2hr.To the blunt end reagent is that every micrograms of DNA adds MgCl 25mmol/ μ g, Tris-HCl 20-25mmol/ μ g, DTT5-10mmol/ μ g, BSA 180-220mg/ μ g, ATP 0.5-0.75mmol and 400-450 unit.T4 DNA links enzyme, sews up 1hr-2hr under the reaction room temperature.
(6), the DNA agarose connects
With agarose dissolving (65 ℃), when reducing to 37 ℃, add and sew up damping fluid, room temperature is sewed up 1hr.
(7), DNA purifying and agarose DNA purifying
Handle according to Millipore ultrafree-probind filter Unit respectively, or handle with Bio-Spin 6 Column posts.
(8), change the bacterium method
1, the preparation of E.coli cell
Inoculation Bacillus coli cells BL21 34 ℃ of shaking culture in aseptic LB substratum are worked as OD 600Be 0.5 o'clock, place ice bath 10min, centrifugation then (10min under the 6000r/min), the cell harvesting thing is suspended in 0.1MMgCl again 2Among the ice bath 40min, centrifugal collection adds the 100mM CaCl that ice bath is crossed in the cell harvesting thing in the solution 22ml, under the aseptic condition, ice bath is cultivated 24hr, and cell is assigned to-70 ℃ of preservations in the little centrifuge tube.
2, E.coli transforms
With cell thawing and ice bath, per 50 μ l cells add 1 μ g DNA, ice bath 30 minutes, and 42 ℃ of water-baths are 2 minutes then, add 1ml LB substratum, change mixture over to 34 ℃ of shaking culture 2hr.The conversion product of this moment 10% contains antibiotic.
(9), protein expression analysis
The 10%LB nutrient solution of packing in shaking bottle adds 5% kantlex in 34 ℃ of overnight incubation, works as OD 600Reach at 1 o'clock, temperature is transferred to 42 ℃ cultivate ice bath behind the 2hr, use in order to the experiment of immunity seal mark.
(10), immunoblot experiment
According to the protein electrophoresis experimental arrangement, gel one dimension electrophoresis is transformed into two-dimensional gel electrophoresis, cultivate through milk then, carry out immunoblot experiment with mouse antibodies IgG and phosphoserine antibody PSR-45.
(11), analysis of amino acids
Use amino acidanalyser, sample is through 6N HCL hydrolysis.
(12), dna sequence analysis
Use the dna sequence analysis instrument
(13), amino acid sequence analysis
Use the amino acid sequence analysis instrument
Two, the fermentation culture of engineering bacteria and inducing
Fermentation condition
34 ℃ of culture temperature, pH7.0 dissolved oxygen DO 20%, air flow quantity is 1VVM-4VVM, mixing speed 450r/min-1000r/min, inoculum size 5%, seed culture medium, LB substratum, kantlex 0.5mg/L, glucose 5g/L.
Stream adds strategy: be advisable with glucose concn control 1.5%, be controlled to be 40% with the DO amount and be advisable.
Work as OD 600Value be culture temperature to be adjusted to 42 ℃ at 100 o'clock to induce 2 hours harvested cells.
Fermention medium is:
Yeast powder 10-30g/L glucose 15-38g/L
NaHPO 4 2.5-8.7g/L (NH 4) 2SO 4 3.6-5.6g/L
FeCl 3 0.8-1.6g/L CoCl 2.6H 2o 0.1-0.2g/L
ZnSO 4 0.1-0.3g/L
The preferred plan of fermention medium is:
Yeast powder 20g/L, glucose 30g/L
NaHPO 4 6.7g/L (NH 4) 2SO 4 5.6g/L
Trace element FeCl 31.2g/L CoCl 2.6H 2O 0.2g/L
ZnSO 4 0.2g/L
Supplemented medium: 10 times of spissated fermention mediums
Three, the purification of Human-like Collagen
Separation purifying technique
Centrifugal collection → cell homogenates → damping fluid extracting → saltout → ion-exchange → column chromatography is made with extra care → lyophilize
Cell add after centrifugal damping fluid (Tis pH8.0,10mM EDTA) by 5 times to wet cell weight with the damping fluid volume.Behind the ultrasonication 20min, with the formic acid damping fluid saltout slightly carry after, spent ion exchange resin adsorbs with behind 0.5M NaCl, the 20mM Tris pH8.0 wash-out, again with golden from chelate column absorption, wash-out, collection protein frozen drying.
The present invention carries out certain heavy with a fragment gene of the collagen protein of human body known array after modified then is cloned in the intestinal bacteria again, the quick breeding of nationality cell is under given conditions by inducing acquisition, and, make its product structure and performance belong to initiative at home, outward through improvement water-soluble to it, that immunity rejects characteristics such as property, stability, one-tenth colloidality, absorptivity.This invention has obtained the high expression level of collagen protein, its expression amount is 20-50%, the Human-like Collagen proline(Pro) of its generation is than the few 40%-60% of animal body collagen protein, this has increased the advantage that a lot of animal body collagen proteins do not possess to the Human-like Collagen that the present invention produces, its chemically reactive amino acid position is more, its derivative kind is variation more, and its performance will more can satisfy the demand of different industries.
The Human-like Collagen that this technology produced not only has animal body collagen protein inherent bio-compatibility and biological heavy absorptivity, and has the reversible gelatigenous characteristic of heat under specified temp; Have the advantage of low immune rejection and virus-free hidden danger, it can realize the high expression level of polymer collagen albumen in unicellular; The Human-like Collagen that it produces is a spirane structure, have the unique chemical structure and the performance that are better than the animal body collagen protein, as the reversible one-tenth colloidality of heat, water-soluble, low immune rejection etc., for example, in order to reduce the immune rejection of organism collagen protein, three peptide chains that we use contain the glycine that has simple side chain, L-Ala, Serine, Xie Ansuans etc. also carry out unique sequence and repeat, this tumor-necrosis factor glycoproteins is from the part in the structure of the known collagen protein sequence of organism, improved organism collagen protein---GXY structure (the X here, Y can be any amino acid).The animal body collagen protein is owing to rotation, typing etc. can not reduce heavily dissolving under the molecular weight condition, therefore Human-like Collagen just can be processed into operating sutures artificial skin, film coating, artificial organs coatings etc. also can combine with silver halide, dyestuff etc. and form the coating with good surface viscosity.

Claims (6)

1, a kind of Human-like Collagen is characterized in that: its sequence is as follows
HDP?VVL?QRR?DWE?NPG?VTQ?LNR?HLA?HAH?PPF?ASD?HPM?GAP
GPA?GAP?GPP?GAP?GPA?GPP?GSA?GAP?GPP?GAP?GPA?GPP?GSA
GAP?GPP?GAP?GPA?GPP?GSA?GAP?GPP?GAP?GPA?GPP?GSA?GAP
GPP?GAP?GPA?GPP?GSA?GAP?GPP?GAP?GPA?GPP?GSA?GAP?GPP
GAP?GPA?GPP?GSA?GAP?GPP?GAP?GPA?GPP?GSA?GAP?GPP?GAP
GPA?GPP?GSA?GAP?GPP?GAP?GPA?GPP?GSA?GAP?GPP?GAP?GPA
GPP?GSA?GAP?GPP?GAP?GPA?GPP?GSA?GAP?GPP?GAP?GPA?GPP
GSA?GAP?GPP?GAP?GPA?GPP?GSA?GAP?GPP?GAH?GPA?GAL?GAH
GPA?GPL?GPA?GPP?GSA?GAP?GAH?GPA?GPL?GAH?GPA?GPL?GAH
GPA?GPL?GAH?GPA?GPL?GAP?GPA?GPP?GSA?GAP?GPP?GAP?GPA
GPP?GSA?GAP?GPP?GAP?GPA?GPP?GSA?GAP?GPP?GAP?GPA?GPP
GSA?GAP?GPP?GAP?GPA?GPP?GSA?GAP?GPP?GAP?GPA?GPP?GSA
GAP?GPP?GAP?GPA?GPP?GSA?GAP?GPP?GAP?GPA?GPP?GSA?GAP
GPP?GAP?GPA?GPP?GSA?GAP?GPP?GAP?GPA?GPP?GSA?GAP?GPP
GAH?GPA?GPL?GAH?GPA?GPL?GAH?GPA?GPL?GAH?GPA?GPL?GAP
GPA?GSA?GAP?GPP?GAP?GPA?GPP?GSA?GAP?GPP?GAP?GPA?GPP
GSA?GAP?GPP?GAP?GPA?GPP?GSA?GAP?GPP?GAP?GPA?GPP?GSA
GAP?GPP?GAP?GPA?GPP?GSA?GAP?GPP?GAP?GPA?GPP?GSA?GAP
GPP?GAP?GPA?GPP?GSA?GAP?GPP?GAP?GPA?GPP?GSA?GAP?GPP
GAP?GPA?GPP?GSA?GAP?GPP?GAP?GPA?GPP?GSA?GAP?GPP?GAP
GPA?GPP?GSA?GAP?GPP?GAH?GPA?GPL?GAH?GPA?GPL?GAH?GPA
GPL?GAH?GPA?GPL?GAP?GPA?GPP?GSA?GAP?GPP?GAP?GPA?GPP
GSA?GAP?GPP?GAP?GPA?GPP?GSA?GAP?GPP?GAP?GPA?GPP?GSA
GAP?GPP?GAP?GPA?GPP?GSA?GAP?GPP?GSA?GAP?GPP?GAP?GPA
GPP?GSA?GAP?GPP?GAP?GPA?GPP?GSA?GAP?GPP?GAP?GPA?GPP
GSA?GAP?GPP?GAP?GPA?GPP?GSA?GAP?GPP?GAP?GPA?GPP?GSA
GAP?GPP?GAH?GPA?GPL?GAH?GPA?GPL?GAH?GPA?GPL?GAM?GAP
GAT?GLS?AGA?THG?LVT?CGL，
Described structure is three chains, triple-helix structure, and its length nucleic acid is 1071bp.
2, the production method of Human-like Collagen according to claim 1 is characterized in that:
1), the structure of the engineering bacteria of Human-like Collagen;
2), the fermentation culture of engineering bacteria;
3), Human-like Collagen inducing and expressing;
4), the purification of Human-like Collagen.
3, the production method of Human-like Collagen according to claim 2 is characterized in that: the structure of the engineering bacteria of described Human-like Collagen is as follows:
1), a fragment gene of the known collagen protein sequence of human body extracted and modifies after, specific again repetition connects then, the DNA reorganization;
2), recombinant DNA is transferred to intestinal bacteria.
4, the production method of Human-like Collagen according to claim 2 is characterized in that: the fermention medium of described engineering bacteria is:
Yeast powder 10-30g/L glucose 15-38g/L
NaHPO 4 2.5-8.7g/L (NH 4) 2SO 4 3.6-5.6g/L
FeCl 3 0.8-1.6g/L CoCl 2.6H 2O 0.1-0.3g/L
ZnSO 4 0.1-0.3g/L。
5, the production method of Human-like Collagen according to claim 2 is characterized in that: the fermentation culture conditions of described engineering bacteria is
34 ℃ of culture temperature, pH7.0 dissolved oxygen DO 20%, air flow quantity is 1VVM-4VVM, mixing speed 450r/min-1000r/min, inoculum size 5%, seed culture medium, LB substratum, kantlex 0.5mg/L, glucose 5g/L, be advisable with glucose concn control 1.5%, be controlled to be 40% with the DO amount and be advisable, work as OD 600Value be 100 o'clock harvested cells.
6, the production method of Human-like Collagen according to claim 2 is characterized in that the purification of described Human-like Collagen is as follows:
Cell after the damping fluid extracting is used in high pressure cell homogenate then, after for the first time formic acid or acetate buffer are saltoutd, then carries out the ion exchange resin exchange through centrifugal collection, and refining and remove thermal source again through metal ion-chelant absorption, elaboration goes lyophilize.
CN01106757.8A 2001-02-21 2001-02-21 Human like collagen and production method thereof Expired - Lifetime CN1196712C (en)

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PCT/CN2002/000424 WO2003106494A1 (en) 2001-02-21 2002-06-14 A human collagen-like protein and the method of producing it

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CN111467560B (en) * 2020-05-27 2022-08-05 陕西巨子生物技术有限公司 Medical hemostatic dressing, preparation method and application thereof
CN112316214B (en) * 2020-11-03 2022-09-30 陕西巨子生物技术有限公司 Injectable hydrogel of recombinant collagen and preparation method thereof
CN114539389B (en) * 2022-02-22 2023-01-31 陕西巨子生物技术有限公司 Recombinant collagen and application thereof
CN117229390A (en) * 2023-09-20 2023-12-15 山东义才和锐生物技术有限公司 Preparation method of recombinant III type humanized collagen

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