CN102321658A - Mutation method of recombinant protein of pig somatic cells - Google Patents
Mutation method of recombinant protein of pig somatic cells Download PDFInfo
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- CN102321658A CN102321658A CN201110204153A CN201110204153A CN102321658A CN 102321658 A CN102321658 A CN 102321658A CN 201110204153 A CN201110204153 A CN 201110204153A CN 201110204153 A CN201110204153 A CN 201110204153A CN 102321658 A CN102321658 A CN 102321658A
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Abstract
The invention discloses a mutation method of recombinant protein of pig somatic cells. The method comprises the following steps: constructing recombinant protein of a cell-penetrating peptide exogenous defined factor for the induction of pig somatic cells; extracting and purifying the cell-penetrating peptide exogenous defined factor from bacteria; and adding the defined factor recombinant protein into a nutrient solution of the pig somatic cells and mutating, wherein the recombinant protein of the cell-penetrating peptide exogenous defined factor can enter the pig somatic cells, can gradually form a cell colony with clear colony edge boundary under the culture condition of step cells, each single cell in the cell colony is round, the cell nucleus is greater and occupies high proportion in the cell, and alkaline phosphatase and step cell specific molecular of cell expressions in the colony are marked as Oct 4 and Nanog. The method provided by the invention can confirm that the defined factor recombinant protein can enable the pig somatic cells to mutate in the aspects of form, growing mode, gene expression and the like.
Description
[technical field]
The present invention relates to the cell mutation technical field, relate in particular to a kind of recombinant protein mutafacient system of porcine somatic cell.
[background technology]
Mutagenesis is the focus of modern life science research, and inducing pluripotent stem cells is the man-made new stem cell that is born through mutagenesis.In addition; Through mutagenesis the common machine somatocyte is laterally broken up and be transformed into neurocyte; These mutagenesises can have been lighted the flame of hoping for the mankind's regenerative medicine, and the seed cell that a large amount of mutagenesis form can effectively be treated persistent ailments such as human cancer, degenerative disorders.In addition, adopt this biotherapy can effectively avoid the application of a large amount of chemicalses.Along with the high speed development of science and technology, recombinant protein will become the main force in the mutagenic processes of cell.
[summary of the invention]
The technical problem that the present invention will solve provides a kind of recombinant protein mutafacient system of porcine somatic cell.
In order to solve the problems of the technologies described above, the technical scheme that the present invention adopts is that a kind of recombinant protein mutafacient system of porcine somatic cell may further comprise the steps:
(1) making of recombinant protein
The recombinant protein prokaryotic expression carrier that uses is pET-28a-EGFP, and express cell is the BL21 competent escherichia coli cell; Adopt 9 arginic cell-penetrating peptides to mediate external source respectively and limit gene oct4, sox2, klf4 and myc, the essentially consist form of recombinant protein is that cell-penetrating peptides-external source limits the factor-green fluorescent protein; The prokaryotic expression carrier that will comprise recombinant protein is transformed into the e. coli bl21 competent cell; Bed board, the single bacterium colony of picking; At 37 ℃ of shaking tables; Contain in the bacteria culture medium of kantlex shaking culture and spend the night, the IPTG that adds final concentration then and be 0.1mmol/L induces reorganization protokaryon protein expression; Inducible protein is expressed the back and is collected thalline, is resuspended in the binding buffer liquid of ice bath precooling, and ultrasonic preceding adding final concentration is 1mmol/L PMSF, on ice the ultrasonic degradation bacterium; The cracking supernatant is incorporated with in the chromatography column of His-Bind resin behind 0.45 μ m membrane filtration, with the washing of rinsing damping fluid, and the recombinant protein of elution buffer elution of bound, through 0.22 μ m membrane filtration degerming ,-20 ℃ are frozen subsequent use;
(2) mutagenesis of recombinant protein
When porcine fetus fibroblasts growth converges 80%, go down to posterity, behind the counting with 5 * 10
5Individual cell inoculation is in petridish, and petridish encapsulates with 0.1% gelatin in advance; 2d equivalent respectively adds recombinant protein that described oct4, sox2, klf4 and myc limit the factor simultaneously in the high sugared DMEM nutrient solution of porcine fetus fibroblasts; Be replaced by the high sugared DMEM nutrient solution that contains 4 μ g/ml bFGF and 10U/ml LIF behind the 24h, continue to cultivate 24h; This process is 1 cycle, so repeats 4-6 cycle; 4-6 all after dates are inoculated into porcine fetus fibroblasts on the mouse fetal inoblast feeder layer after with trysinization, and the cell count of each petridish inoculation is about 1 * 10
5, continue to cultivate with the high sugared DMEM nutrient solution that contains 4 μ g/ml bFGF and 10U/ml LIF; Every day, observation of cell changed later on, and 2-3d changes liquid 1 time, observed to have or not typical stem cell-like cell clone to occur.
9 amino acid of above-mentioned cell-penetrating peptides consist of l-arginine-l-arginine-l-arginine-l-arginine-l-arginine-l-arginine-l-arginine-l-arginine-l-arginine.
Albumen is being brought into play important function in the process of growth of cell, the growth of cell, propagation or old and feeble all closely bound up with proteic function.The foreign protein peptide-mediated by cell-penetrating gets into growth and the propagation that can regulate cell behind the cell, thus the biological function of performance foreign protein.The mode of mediate foreign gene expression at present is a lot, virus widespread use with stable, efficient mediate foreign gene.But virus-mediated foreign gene exists the cell of expression alien gene to be prone to the cancerization distortion phenomenon at cell inner expression.Cell-penetrating peptides is made up of 9 or 11 amino-acid residues as a kind of novel polypeptide, is similar to polypeptide product natural in the cell, for get into any cell all have nontoxic with have no side effect.In addition, cell-penetrating peptides portability external source biomacromolecule gets into viable cell, and does not influence entrained proteinic BA; Do not receive simultaneously the influence of cell type; It is fast that its mediation has speed, do not rely on temperature, energy, cytolemma antibody, reaches characteristics such as pair cell does not damage; Cell-penetrating peptides is this to have the unique function of passing through cytolemma, thus mediation foreign protein performance biological function.
The invention has the beneficial effects as follows:
Effectively avoided virus stable integration at random owing to foreign gene in the transgenic process to bring potential cell cancerization distortion harm; Utilize albumen to have the characteristics of no side effects; 9 arginic cell-penetrating peptides carry foreign protein and green fluorescent protein is formed recombinant protein jointly through making up, by intestinal bacteria at short notice High-efficient Production go out the recombinant protein of a large amount of qualification factors.From intestinal bacteria, extract the also recombinant protein of the purifying cells penetrating peptide external source qualification factor; Qualification factor recombinant protein added to carry out mutagenesis in the porcine fetus fibroblasts nutrient solution; The recombinant protein that cell-penetrating peptides carries the external source qualification factor can get in the porcine fetus fibroblasts; Separation and Culture goes out colony edge boundary cell colony clearly gradually under stem cell cultivation conditions, and it is bigger that the individual cells in the cell colony presents nucleus, and proportion is higher in cell; The colony inner cell is expressed SEAP, and stem cell specific molecular marker Oct4 and Nanog protein expression are positive.The present invention confirms to utilize the artificial qualification factor recombinant protein of producing can make porcine fetus fibroblasts at aspect generation mutagenesises such as form, growth pattern, genetic expressions.
[description of drawings]
Below in conjunction with accompanying drawing and embodiment the present invention is done further detailed explanation.
Fig. 1 is the cellular form of embodiment of the invention porcine fetus fibroblasts.
Fig. 2 is the form (right figure is the high power lens enlarged view at left figure square frame place) that forms stem cell colonies after the effect of embodiment of the invention porcine fetus fibroblasts recombinant protein.
[embodiment]
1 materials and methods
1.1 main experiment material
The Recombinant Protein Expression cell is the BL21 competent escherichia coli cell;
9 amino acid of cell-penetrating peptides consist of l-arginine-l-arginine-l-arginine-l-arginine-l-arginine-l-arginine-l-arginine-l-arginine-l-arginine;
The basic recombination structure of recombinant protein prokaryotic expression carrier is that pET-28a-cell-penetrating peptides-external source limits the factor-green fluorescent protein;
Cell culture fluid is high sugared DMEM nutrient solution;
1.2 main working method
1.2.1 the making of recombinant protein
The recombinant protein prokaryotic expression carrier that uses is pET-28a-EGFP, and express cell is the BL21 competent escherichia coli cell.The amino acid of cell-penetrating peptides consists of l-arginine-l-arginine-l-arginine-l-arginine-l-arginine-l-arginine-l-arginine-l-arginine-l-arginine; Adopt 9 arginic cell-penetrating peptides to mediate external source respectively and limit factor oct4, sox2, klf4 and myc, the recombinant protein essentially consist form of structure is that cell-penetrating peptides-external source limits the factor-green fluorescent protein.The prokaryotic expression carrier that will comprise recombinant protein is transformed into the e. coli bl21 competent cell; Bed board, the single bacterium colony of picking; At 37 ℃ of shaking tables; Contain in the bacteria culture medium of kantlex shaking culture and spend the night, the IPTG that adds final concentration then and be 0.1mmol/L induces reorganization protokaryon protein expression, is 3h 22 ℃ of abduction delivering times.After inducible protein was expressed, the centrifugal 10min of 12000rpm collected thalline, is resuspended in the binding buffer liquid of ice bath precooling, and ultrasonic preceding adding final concentration is 1mmol/L PMSF, on ice the ultrasonic degradation bacterium.The cracking supernatant is incorporated with in the chromatography column of His-Bind resin behind 0.45 μ m membrane filtration, with the washing of rinsing damping fluid, and the recombinant protein of elution buffer elution of bound, through 0.22 μ m membrane filtration degerming ,-20 ℃ are frozen subsequent use.
1.2.2 the mutagenesis of recombinant protein
When porcine fetus fibroblasts growth converges 80%, go down to posterity, behind the counting with 5 * 10
5Individual cell inoculation is in the 60mm petridish, and the 60mm ware shifts to an earlier date 15min and encapsulates with 0.1% gelatin.2d while equivalent respectively adds oct4, sox2, klf4 and four kinds of recombinant proteins that limit the factor of myc in the sugared DMEM nutrient solution of height; Every kind of concentration that limits factor recombinant protein is 10 μ g/ml; Be replaced by the high sugared DMEM nutrient solution that contains 4 μ g/ml bFGF and 10U/mlLIF behind the 24h, continue to cultivate 24h.The oct4 of equivalent adding simultaneously, sox2, klf4 and four kinds of recombinant proteins that limit the factor of myc are in nutrient solution respectively behind the 24h, and this operating process is 1 mutagenic treatment cycle, so repetitive operation 4-6 mutagenic treatment cycle.At 4-6 mutagenic treatment week after date, porcine fetus fibroblasts is inoculated on the mouse fetal inoblast feeder layer after with trysinization, the cell count of each 60mm ware inoculation is about 1 * 10
5, continue to cultivate with the high sugared DMEM nutrient solution that contains 4 μ g/ml bFGF and 10U/ml LIF.Every day, observation of cell changed later on, and 2-3d changes liquid 1 time, observed to have or not typical stem cell-like cell clone to occur.
1.2.3 the evaluation of mutagenized cell
Mutagenized cell is fixed through 4% Paraformaldehyde 96, and 1% bovine serum albumin (BSA) room temperature sealing 30 minutes is added an anti-anti-Oct4 of rabbit of being, goat anti Nanog by 1: 200 back 4 ℃ of incubated overnight of dilution.PBS washes 3 times, and two of CY3 red fluorescence mark anti-is hatched 1h by 1: 200 dilution back room temperature lucifuge, and PBS washes 3 times, carries out the nucleus observations under the fluoroscope that dyes behind the 1min with the DAPI of 1 μ g/ml.
With 4% Paraformaldehyde 96 fixed cell, add the SEAP dye liquor and dye, be colored as reddish-brown or coffee-like particle is positive with endochylema.
2 results
2.1 the stem-like cell colony of mutagenized cell forms
Inoculation 1 * 10
5Individual porcine fetus fibroblasts is in the 60mm petridish, and 2d adds qualification factor recombinant protein, and every kind of concentration that limits factor recombinant protein is 10 μ g/ml.Change the fresh high sugared DMEM nutrient solution that contains 4 μ g/mlbFGF and 10U/ml LIF behind the 24h into; Induce 4-6 all after date; Then cell is inoculated on the mouse fetal inoblast feeder layer after with tryptic digestion, continues with the high sugared DMEM nutrient solution cultivation that contains 4 μ g/ml bFGF and 10U/ml LIF.The form change appearred in the part porcine fetus fibroblasts in the porcine fetus fibroblasts (Fig. 1) of the fibrous form of fusiformis after adding 3-4 cycle recombinant protein; No longer show as the fibrous form of typical fusiformis; But the porcine fetus fibroblasts digestive inoculation is continued to cultivate to feeder layer, and no stem cell-like cell colony formed after 20d was cultivated in continuation.Increase to limit the processing cycle of factor recombinant protein, through 6 handle all after dates again with the porcine fetus fibroblasts digestive inoculation to mouse fetal inoblast feeder layer, having a small amount of clear-cut stem cell-like cell colony to form gradually continuing on the feeder layer to cultivate about 10-15d; It is rounded mainly to show as the interior composition of cell colony cell; Round cell closely connects, and iuntercellular is assembled the formation cell mass each other, and the nucleus of individual cells is bigger in the colony; Colony protrudes in the feeder layer surface and is nest like (Fig. 2); The cell colony growth is rapid, and every 2-3d goes down to posterity once, and it is stable that the cell growth state after going down to posterity keeps.
2.2 the stem cell labeling of mutagenized cell is expressed
Oct4 and Nanog are the stem cell specific molecular marker; Use the specific antibody of Oct4 and Nanog that mutagenized cell is carried out the immunocytochemistry check and analysis; Immunofluorescence dyeing shows cell expressing Oct4 and the Nanog albumen that mutagenesis takes place; These specific anti body proteins mainly are positioned nucleus; Consistent with the proteic coloration result of appraising and deciding position and nuclear specificity dyestuff DAPI of Oct4 and Nanog, mutagenized cell is expressed stem cell specific marker Oct4 and Nanog, and porcine fetus fibroblasts is not expressed stem cell specific marker Oct4 and Nanog before the mutagenesis.In addition, the cell colony SEAP of mutagenesis detects and is strong positive, and the whole cell colony color after the dyeing is a red-purple, and the porcine fetus fibroblasts SEAP detects negative before the mutagenesis.Detected result shows through the mutagenesis porcine fetus fibroblasts of recombinant protein can demonstrate characteristic change at aspects such as the form of cell, growth pattern, genetic expressions.
Claims (2)
1. the recombinant protein mutafacient system of a porcine somatic cell is characterized in that, may further comprise the steps:
(1) making of recombinant protein
The recombinant protein prokaryotic expression carrier that uses is pET-28a-EGFP, and express cell is the BL21 competent escherichia coli cell; Adopt 9 arginic cell-penetrating peptides to mediate external source respectively and limit gene oct4, sox2, klf4 and myc, the essentially consist form of recombinant protein is that cell-penetrating peptides-external source limits the factor-green fluorescent protein; The prokaryotic expression carrier that will comprise recombinant protein is transformed into the e. coli bl21 competent cell; Bed board, the single bacterium colony of picking; At 37 ℃ of shaking tables; Contain in the bacteria culture medium of kantlex shaking culture and spend the night, the IPTG that adds final concentration then and be 0.1mmol/L induces reorganization protokaryon protein expression; Inducible protein is expressed the back and is collected thalline, is resuspended in the binding buffer liquid of ice bath precooling, and ultrasonic preceding adding final concentration is 1mmol/L PMSF, on ice the ultrasonic degradation bacterium; The cracking supernatant is incorporated with in the chromatography column of His-Bind resin behind 0.45 μ m membrane filtration, with the washing of rinsing damping fluid, and the recombinant protein of elution buffer elution of bound, through 0.22 μ m membrane filtration degerming ,-20 ℃ are frozen subsequent use;
(2) mutagenesis of recombinant protein
When porcine fetus fibroblasts growth converges 80%, go down to posterity, behind the counting with 5 * 10
5Individual cell inoculation is in petridish, and petridish encapsulates with 0.1% gelatin in advance; 2d equivalent adds the recombinant protein of described oct4, sox2, klf4 and the myc qualification factor simultaneously in the high sugared DMEM nutrient solution of porcine fetus fibroblasts; Be replaced by the high sugared DMEM nutrient solution that contains 4 μ g/ml bFGF and 10U/ml LIF behind the 24h, continue to cultivate 24h; This process is 1 cycle, so repeats 4-6 cycle; 4-6 all after date is inoculated into porcine fetus fibroblasts on the mouse fetal inoblast feeder layer with trysinization, and the cell count of each petridish inoculation is about 1 * 10
5, continue to cultivate with the high sugared DMEM nutrient solution that contains 4 μ g/ml bFGF and 10U/ml LIF; Every day, observation of cell changed later on, and 2-3d changes liquid 1 time, observed to have or not typical stem-like cell clone to occur.
2. the recombinant protein mutafacient system of porcine somatic cell according to claim 1 is characterized in that, 9 amino acid of described cell-penetrating peptides consist of l-arginine-l-arginine-l-arginine-l-arginine-l-arginine-l-arginine-l-arginine-l-arginine-l-arginine.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105531284A (en) * | 2013-07-12 | 2016-04-27 | 杰姆维克斯&凯尔有限公司 | Cell-penetrating peptide and conjugate comprising same |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010115052A2 (en) * | 2009-04-03 | 2010-10-07 | The Mclean Hospital Corporation | Induced pluripotent stem cells |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2010115052A2 (en) * | 2009-04-03 | 2010-10-07 | The Mclean Hospital Corporation | Induced pluripotent stem cells |
Non-Patent Citations (2)
| Title |
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| 殷慧群: "猪诱导性多潜能干细胞的建立", 《中国优秀博士论文数据库 农业科技辑》 * |
| 殷慧群等: "限定因子诱导胎猪成纤维细胞重编程为多能性细胞", 《生物化学与生物物理进展》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105531284A (en) * | 2013-07-12 | 2016-04-27 | 杰姆维克斯&凯尔有限公司 | Cell-penetrating peptide and conjugate comprising same |
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Application publication date: 20120118 |