WO2006111060A1 - Procede permettant d’isoler et de purifier un polypeptide immuno-modulant a partir du placenta de vache - Google Patents
Procede permettant d’isoler et de purifier un polypeptide immuno-modulant a partir du placenta de vache Download PDFInfo
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- WO2006111060A1 WO2006111060A1 PCT/CN2006/000219 CN2006000219W WO2006111060A1 WO 2006111060 A1 WO2006111060 A1 WO 2006111060A1 CN 2006000219 W CN2006000219 W CN 2006000219W WO 2006111060 A1 WO2006111060 A1 WO 2006111060A1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/48—Reproductive organs
- A61K35/50—Placenta; Placental stem cells; Amniotic fluid; Amnion; Amniotic stem cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
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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
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4715—Pregnancy proteins, e.g. placenta proteins, alpha-feto-protein, pregnancy specific beta glycoprotein
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the invention relates to the technical field of biological and medical extraction. More specifically, the present invention relates to a method for isolating and purifying an immunomodulatory active polypeptide from a bovine placenta.
- placental immunoregulators The study of placental immunoregulators is the first active ingredient in China to be extracted from the placenta of healthy women.
- Liu Yuexin a new immunomodulator - preparation and research of placental factors, "Chinese Journal of Immunology", 1985, 1 (5)) first reported the use of "homogenation - dialysis” method from healthy women A small molecule active substance extracted from the placenta, and this factor is called placental factor; 1994, Huang Chuhua et al.
- the tissue mincer was made into a placental tissue homogenate.
- the homogenate was frozen in a refrigerator at -20 °C for more than 48 hours.
- placental immunoregulators are primarily small molecule polypeptides.
- placental immunoregulators are safe, non-toxic, non-antigenic, and long-term use does not reduce the activity due to antibody production. It is an efficient and safe immunomodulator and enhancer.
- the current research on placental immunoregulators mainly focuses on function and application, but there are few reports on the separation and purification of its main active components.
- bovine placenta As a new type of placenta resource, bovine placenta has similarities with human placenta in terms of its source, structure and composition, but there are also significant differences. Therefore, with reference to the study of human placental immunoregulatory factor, it is of great theoretical and technical significance to separate and purify immunoregulatory factors in bovine placenta by different separation and purification techniques.
- Another object of the present invention is to provide an immunomodulatory active polypeptide extracted from bovine placenta
- ion exchange chromatography is carried out in a system in which an ion exchanger is used as a stationary phase and a liquid is a mobile phase.
- the ion exchanger is composed of a matrix, a charge group, and a counter ion.
- the reaction of the ion exchanger with the ion or ionic compound in the aqueous solution is carried out mainly by ion exchange or by adsorption of ions or ionic compounds in the solution by means of a charge group on the ion exchanger.
- Ion exchange chromatography is based on the difference in the charged state (or polarity) of various materials.
- the appropriate type of ion exchanger must be selected according to the dissociation properties of the substances it contains, and the adsorption and elution conditions must be controlled, mainly the ionic strength and pH of the eluent, so that the components in the mixture are in affinity.
- the sequence is sequentially eluted from the column.
- Gel exclusion chromatography allows a sample solution containing various molecules to flow slowly through a gel column, with each molecule performing simultaneous vertical downward movement and non-directional diffusion motion within the column. Because of its large molecular diameter, macromolecular substances are not easy to enter the micropores of gel particles, but can only be distributed between particles, so they move faster when eluting; while small molecules are in the gap of gel particles. In addition to diffusion, it also enters the gel phase. During the downward movement, it diffuses from the gel particles into the particle gap and then enters the other gel particles, thus continuously achieving the entry and diffusion processes, thus the small molecular substances.
- the downward movement speed is lower than that of the macromolecular substance, so that the macromolecule, the medium molecule, and the small molecule in the sample flow out of the column, respectively, thereby achieving separation of various molecules thereof.
- the outstanding advantage of gel chromatography is that the gel used in chromatography is an inert carrier, has no charge, weak adsorption, mild operating conditions, can be carried out over a wide temperature range, does not require organic solvents, and separates components. The maintenance of physical and chemical properties is unique.
- Reversed phase chromatography is a very widely used method of separation.
- the primary effect of solute retention is hydrophobic.
- the so-called hydrophobic action means that when a non-polar solute is present in the water, the interaction between the solute molecules, the interaction between the solute molecules and the water molecules is much smaller than the interaction between the water molecules, and thus the solute molecules are easily removed from the water. Therefore, the residence time of different compounds in the column is different because of their different hydrophobic properties, so that they are separated from each other.
- the inventors have creatively combined these three separation methods through a large number of experimental studies, that is, the combination of anion exchange chromatography, gel exclusion chromatography and reversed-phase high performance liquid chromatography to realize the method from the cattle placenta.
- the immunomodulatory active polypeptide is extracted, and the molecular weight, isoelectric point and sequence of the isolated and purified immunomodulatory active polypeptide are determined.
- the present invention relates to an immunomodulatory functional polypeptide isolated from a bovine placenta.
- Immunomodulation The functionally active polypeptide is characterized by: The molecular weight of the immunomodulatory active polypeptide measured by MALDI-TOF-MS (matrix-assisted laser desorption time-of-flight mass spectrometry) is 2133.52 Da, and the isoelectricity measured by CIEF (capillary isoelectric focusing) The point was 3.82, and the sequence was determined by Tyr-X-Phe-Leu-Gly-Leu-Pro-Gly-X-Thr using a Type 491 protein sequencer (Applied Biosystems, USA), where X is an amino acid.
- the invention also relates to a method of preparing an immunomodulatory functional polypeptide isolated from a bovine placenta.
- the method comprises taking fresh bovine placenta, washing the diced block, adding twice (weight/volume) phosphate buffer pH 6.8-7.5, homogenizing, centrifugation at 12000 r/m, sedimentation, and taking it, as is well known to those skilled in the art.
- the anion exchange chromatography, gel exclusion chromatography, and reversed-phase high performance liquid chromatography separation steps of the salt buffer solution are performed by RP-HPLC and capillary electrophoresis on the active components collected by reversed-phase high performance liquid chromatography. All of them were single peaks, and the molecular weight of the immunomodulatory active polypeptide was determined by MALDI-TOF-MS.
- the isoelectric point of the immunomodulatory active polypeptide was determined by CIEF, and the immunomodulatory active polypeptide sequence was determined by a protein sequencer.
- the anion exchange chromatography is performed by dissolving the lyophilized powder in a weight ratio of 4-10:1 by 10-30 mmol/L of pH 6.8-7.5 phosphate buffer.
- the solution is applied as a sample solution, and is applied to the anion exchange column at a flow rate of 0.5-1.5 mL/min, and then eluted with a gradient at a flow rate of 0.5 - 1.5 mlJmin; the eluent A is 10-30 mmol/L of phosphate.
- Buffer, B solution is added with 0.5-1.5mol/L sodium chloride solution in solution A.
- Gradient method is: 0-600 minA solution; 600-1000 min B solution 0-100%, in vitro cultured lymphocyte proliferation experiment
- the MTT method thiazole method detects, screens, and collects components containing an immunomodulatory active polypeptide after anion exchange chromatography.
- the anion exchange column has a length to diameter ratio of 8-18, and the sample volume can reach 25 % -30% of the bed volume.
- the weight ratio of the lyophilized powder to the buffer for dissolving the lyophilized powder is from 4 to 8:1. More preferably, the weight ratio of the lyophilized powder to the buffer for dissolving the lyophilized powder is 5-8:1.
- the buffer used to dissolve the lyophilized powder in the method of the invention may be, for example, one or more selected from the group consisting of Na 2 HP0 4 -NaH 2 P0 4 , K 2 HP0 4 -KH 2 P0 4 , K 2 HP0 4 - Buffer in NaH 2 P0 4 or Na 2 HP0 4 -KH 2 P0 4 .
- the solution of the dissolved lyophilized powder is a buffer of 10-30 mmd/L Na 2 HP0 4 - NaH 2 P0 4 pH 6.8-7.5.
- the eluent A used in the anion exchange chromatography of the method of the present invention may be, for example, one or more selected from the group consisting of Na 2 HP0 4 -Na3 ⁇ 4P0 4 , K 2 HP0 4 -KH 2 P0 4 , K 2 HP0 4 - Eluent in NaH 2 P0 4 or Na 2 HP04-KH 2 P0 4 .
- the gel exclusion chromatography is to directly load the component containing the immunomodulatory active polypeptide collected by the anion exchange chromatography at a flow rate of 8-12 mL/h.
- the washing phase was selected from 10-30 mmol/L phosphate pH 6.8-7.5 buffer, eluted at a flow rate of 8-12 mL/h, and the lymphocyte proliferation experiment was carried out in vitro.
- the MTT method detects, screens, and collects components containing an immunomodulatory active polypeptide after anion exchange chromatography.
- the gel exclusion chromatography column has a length to diameter ratio of 60-90, and the sample volume can reach 1% -10% of the bed volume.
- the mobile phase used in the gel exclusion chromatography is one or more selected from the group consisting of
- the reversed-phase high performance liquid chromatography is to directly load a fraction containing an immunomodulatory active polypeptide collected by gel exclusion chromatography at a flow rate of 0.8 - 1.2 mL/min.
- the washing phase A solution is selected from 5-10% acetonitrile with 0.01-0.1% trifluoroacetic acid
- the B solution is selected from 40-60% acetonitrile with 0.01-0.1% trifluoroacetic acid.
- the gradient was eluted with a gradient of 0.8-1.2 mL/min. The gradient was: 0-8 min A solution; 8- 12 min B solution 0-100%; 12-16 min B solution.
- the reversed-phase high performance liquid chromatography has a ratio of length to diameter of 25-50, and the sample volume can reach 1% -10% of the volume of the bed.
- the washing liquid phase A is selected from the group consisting of 0.02-0.08% trifluoroacetic acid 5-8% acetonitrile, and the B liquid is selected from 0.02-0.08% trifluoroacetic acid 40-60% acetonitrile.
- the anion exchange column is agarose DEAE Sepharose CL-6B, DEAE-Sepharose FF or diethylaminoethyl-bound dextran combined with diethylaminoethyl.
- the anion exchange column is an anion exchange column containing DEAE Sepharose CL-6B anion exchange medium, DEAE-Sepharose FF, DEAE-Sephadex A-25.
- the gel exclusion chromatography column is an inert porous network material containing dextran Sephadex G-25, Sephadex G-50 or polyacrylamide gel Bio-gel- P-4, Bio-gel-P-6 or Bio-gel-P-10, a gel exclusion chromatography column that separates substances in a protein mixture by molecular size.
- the gel exclusion chromatography column is a gel exclusion device containing Sephadex G-25, Sephadex G-50, Bio-gel-P-4 or Bio-gel-P-6 medium. Column.
- the reversed-phase high performance liquid chromatography column is equipped with ODS.
- ODS (18-mercapto-silica) reverse-phase packing Sephasil peptide C 18 , Polymer C 18 in a reversed-phase high performance liquid chromatography column.
- the immunomodulatory active polypeptide having a purity of more than 90% can be obtained by the method of the present invention, and thus the biological activity can reach the medicinal standard.
- the immunomodulatory active polypeptide obtained by the method of the present invention provides a rich raw material for the development of a health food having immunomodulatory activity.
- the yield of the obtained immunomodulatory active polypeptide obtained by the method of the present invention is 1% based on the dry basis of lg immunomodulatory active polypeptide OOg calf placenta.
- FIG 1 is a 3 ⁇ 4 immunomodulatory activity of the polypeptide in the exchange column anionic DEAE Sepharose CL-6B of the embodiment of the present invention, the separation pattern;
- Peak la is a component containing an immunomodulatory active polypeptide
- FIG. 2 is a bar graph of a lymphocyte proliferation experiment in vitro of four components obtained by immunomodulating active polypeptide on a DEAE Sepharose CL-6B anion exchange column according to an embodiment of the present invention
- Component la is a component containing an immunomodulatory active polypeptide
- Figure 3 is a diagram showing the separation of immunomodulatory active polypeptides on a gel exclusion chromatography Sephadex G-25 column in the examples of the present invention
- Peak lb is a component containing an immunomodulatory active polypeptide
- Component lb is a component containing an immunomodulatory active polypeptide
- Figure 5 is an isolated map of an immunomodulatory active polypeptide in a reverse phase high performance liquid chromatography Sephasil peptide (3 18 ) in an embodiment of the invention.
- Peak 4 is a component containing an immunomodulatory active polypeptide.
- 6 is a lymphocyte proliferation experiment of in vitro culture of 7 components obtained by the immunomodulatory active polypeptide in the embodiment of the present invention on Sephasil peptide C 18 ;
- Component 4 is a component containing an immunomodulatory active polypeptide.
- Figure 7 is a purity identification map of the immunomodulatory active polypeptide on Sephasil peptide C 18 in the embodiment of the present invention, and the immunomodulatory active polypeptide component is more than 90%.
- Figure 8 is a chromatogram of the purity of an immunomodulatory active polypeptide in capillary electrophoresis according to an embodiment of the present invention. ⁇ detailed description ⁇
- mice The spleens of the mice were taken out under aseptic conditions, lymphocytes were separated by lymphocyte separation solution, and the cell concentration was adjusted to lxl0 6 /mL with complete 1640 medium.
- control group and the experimental group were set up respectively to test the ability of each component to promote the proliferation of spleen lymphocytes.
- a mouse spleen lymphocyte suspension (lxl0 6 /mL) 100 L was added to a 96-well culture plate.
- the control group was added with physiological saline 100 and the experimental group was each added with 100 ⁇ M.
- MTT 20 L was added to each well under aseptic conditions, gently mixed, and culture was continued for 4 hours. Then, dimethyl sulfoxide 100 was added to each well for 10 minutes at room temperature, and the OD value of each well was measured by ELISA at 570 nm.
- the P value of the experimental group was calculated by the analysis of variance, and the t test was used for the significant analysis.
- MALDI-TOF-MS measures the molecular weight of immunomodulatory active polypeptides.
- CIEF measures the isoelectric point of the immunomodulatory active polypeptide.
- a protein sequencer measures the immunomodulatory active polypeptide sequence.
- fraction 1 containing the immunomodulatory active polypeptide collected by the above anion exchange chromatography was directly loaded onto a 1.0 x 75 cm Sephadex G-25 gel exclusion chromatography column at a flow rate of 8 mL/h.
- the washing phase was eluted with isocratic using 20 mmol/L Na 2 HP0 4 - Na 3 ⁇ 4 P0 4 buffer, pH 7.4.
- the fraction lb containing the immunomodulatory active polypeptide collected by the gel exclusion chromatography described above was directly loaded onto a Sephasil peptide 18 reversed-phase high performance liquid chromatography column at a flow rate of 1 mL/min.
- the washing phase A 10% acetonitrile with 0.05% trifluoroacetic acid was used, and the liquid B was treated with 60% acetonitrile added with 0.05% trifluoroacetic acid. Elution with a gradient.
- the gradient method is:
- Fig. 5 The results of reversed-phase high performance liquid chromatography elution are shown in Fig. 5.
- the in vitro cultured lymphocyte proliferation assay was used for activity detection.
- the experimental results are shown in Fig. 6. It can be seen from the results of the activity detection experiment that the component 4 obtained by reversed-phase high performance liquid chromatography is a higher purity immunomodulatory active polypeptide.
- Component 4 was further subjected to nanofiltration desalting and lyophilization to obtain an immunomodulatory active polypeptide product.
- the purity of the immunomodulatory active polypeptide product was determined on Sephasil peptide C 18 and capillary electrophoresis. The maps are shown in Figure 7 and Figure 8.
- the final purified fraction was subjected to RP-HPLC and CE detection, all of which were single peaks.
- the molecular weight of the immunomodulatory active polypeptide was determined to be 2133.52 Da by MALDI-TOF-MS.
- the isoelectric point of the immunomodulatory active polypeptide was determined by CIEF to be 3.82.
- the sequence of the immunomodulatory active polypeptide was determined using a Type 491 protein sequencer:
- fraction 1 containing the immunomodulatory active polypeptide collected by the above anion exchange chromatography was directly loaded onto a 1.0 x 75 cm Sephadex G-25 gel exclusion chromatography column at a flow rate of 10 mL/h.
- the washing phase was eluted with isocratic using 20 mmol/L Na 2 HP0 4 -NaH 2 P0 4 buffer at pH 6.8.
- the fraction of the immunomodulatory active polypeptide-containing fraction collected by the above gel exclusion chromatography is directly
- the sample was applied to a Sephasil peptide C 18 reversed-phase high performance liquid chromatography column at a flow rate of 1 mL/min.
- the washing phase A solution was selected from 8% acetonitrile added with 0.05% trifluoroacetic acid, and the mash was selected to be 55% acetonitrile added with 0.05% trifluoroacetic acid. Elution with a gradient.
- the gradient method is:
- Component 4 was further subjected to nanofiltration desalting and lyophilization to obtain an immunomodulatory active polypeptide product.
- the purity of the immunomodulatory active polypeptide product was determined on Sephasil peptide C 18 and capillary electrophoresis, and the map was similar to that of Figures 7 and 8.
- the final purified fraction was subjected to RP-HPLC and CE detection, all of which were single peaks.
- the molecular weight of the immunomodulatory active polypeptide was determined to be 2133.52 Da by MALDI-TOF-MS.
- the isoelectric point of the immunomodulatory active polypeptide was determined by CIEF to be 3.82.
- the sequence of the immunomodulatory active polypeptide was determined using a Type 491 protein sequencer: Tyr-X-Phe-Leu-Gly-Leu-Pro-Gly-X-Thr (X-amino acid).
- the immunomodulatory active polypeptide-containing fraction la collected by the above anion exchange chromatography was directly loaded onto a 1.0 x 75 cm Sephadex G-25 gel exclusion chromatography column at a flow rate of 12 mL/h.
- the washing phase was eluted with isothermality using 20 mmol/L Na 2 HP0 4 - Na 3 ⁇ 4 P0 4 buffer at pH 7.2.
- the gel elution chromatography elution results and the activity assay of the in vitro cultured lymphocyte proliferation assay are similar to those in Fig. 3 and Fig. 4.
- the fraction obtained by gel exclusion chromatography contains an immunomodulatory active polypeptide (a peak indicated by an arrow in the figure).
- the fraction lb containing the immunomodulatory active polypeptide collected by the gel exclusion chromatography described above was directly loaded onto a Sephasil column 18 reversed-phase high performance liquid chromatography column at a flow rate of 1 mL/min.
- the washing phase A was selected to be 5% acetonitrile added with 0.05% trifluoroacetic acid, and the mash was selected to be 45% acetonitrile added with 0.05% trifluoroacetic acid.
- the gradient method is:
- Component 4 was further subjected to nanofiltration desalting and lyophilization to obtain an immunomodulatory active polypeptide product.
- the purity of the immunomodulatory active polypeptide product was determined on Sephsil peptide C 18 and capillary electrophoresis, and the map was similar to that of Figs. 7 and 8.
- the final purified fraction was subjected to RP-HPLC and CE detection, all of which were single peaks.
- the molecular weight of the immunomodulatory active polypeptide was determined to be 2133.52 Da by MALDI-TOF-MS.
- the isoelectric point of the immunomodulatory active polypeptide was determined by CIEF to be 3.82.
- the sequence of the immunomodulatory active polypeptide was determined by a Type 491 protein sequencer: Tyr-X-Phe-Leu-Gly-Leu-Pro-Gly-X-Thr (X-amino acid).
- Example 4 The sequence of the immunomodulatory active polypeptide was determined by a Type 491 protein sequencer: Tyr-X-Phe-Leu-Gly-Leu-Pro-Gly-X-Thr (X-amino acid).
- placenta processing Take fresh bovine placenta, wash and cut, add twice (w/v) pH 7.0 phosphate buffer, homogenate, centrifuge at 12000r/m, precipitate, take supernatant, 10,000 molecular weight cut-off ultrafiltration, aromatic polyamide
- the nanofiltration membrane is desalted and freeze-dried to obtain a sample lyophilized powder containing the immunomodulatory active polypeptide;
- the anion exchange chromatography elution results and the activity detection results using the in vitro cultured lymphocyte proliferation assay were similar to those of Figs. 1 and 2, respectively.
- the components obtained by ion exchange chromatography contained the immunomodulatory active polypeptide 1 (the peak indicated by an arrow in the figure).
- the immunomodulatory active polypeptide-containing fraction la collected by the above anion exchange chromatography was directly loaded onto a 1.0 x 75 cm Sephadex G-25 gel exclusion chromatography column at a flow rate of 12 mL/h.
- the washing phase was eluted with isocratic using 20 mmol/L Na 2 HP0 4 -NaH 2 P0 4 buffer at pH 7.2.
- the gel elution chromatography elution results and the activity assay of the in vitro cultured lymphocyte proliferation assay are similar to those in Fig. 3 and Fig. 4.
- the fraction obtained by gel exclusion chromatography contains an immunomodulatory active polypeptide (a peak indicated by an arrow in the figure).
- the fraction lb containing the immunomodulatory active polypeptide collected by the gel exclusion chromatography described above was directly loaded onto a Sephasil column 18 reversed-phase high performance liquid chromatography column at a flow rate of 1 mL/min.
- the washing phase A 5% acetonitrile with 0.05% trifluoroacetic acid was used, and the mash was selected to be 60% acetonitrile with 0.05% trifluoroacetic acid. Elution with a gradient.
- the gradient method is:
- identification and analysis Component 4 was further subjected to nanofiltration desalting and lyophilization to obtain an immunomodulatory active polypeptide product.
- the purity of the immunomodulatory active polypeptide product was determined on Sephasii peptide C 18 and by capillary electrophoresis, and the map was similar to that of Figures 7 and 8.
- the final purified fraction was subjected to RP-HPLC and CE detection, all of which were single peaks.
- the molecular weight of the immunomodulatory active polypeptide was determined to be 2133.52 Da by MALDI-TOF-MS.
- the isoelectric point of the immunomodulatory active polypeptide was determined by CIEF to be 3.82.
- the sequence of the immunomodulatory active polypeptide was determined using a Type 491 protein sequencer: Tyr-X-Phe-Leu-Gly-Leu-Pro-Gly-X-Thr (X-amino acid).
- the elution results of anion exchange chromatography were similar to those of Figure 1.
- the activity of the lymphocyte proliferation test was carried out in vitro, and the detection result was similar to that of Fig. 2. It can be seen from the results of the activity test that the components obtained by ion exchange chromatography contain the immunomodulatory active polypeptide 1 (the peak indicated by the arrow in the figure) ).
- fraction 1 containing the immunomodulatory active polypeptide collected by the above anion exchange chromatography was directly loaded onto a 1.0 x 75 cm Sephadex G-25 gel exclusion chromatography column at a flow rate of 10 mL/h.
- the washing phase was eluted with isocratic using 20 mmol/L Na 2 HP0 4 - Na 3 ⁇ 4 P0 4 buffer, pH 7.4.
- Component 4 was further subjected to nanofiltration desalting and lyophilization to obtain an immunomodulatory active polypeptide product.
- the purity of the immunomodulatory active polypeptide product was determined on Sephasil peptide C 18 and capillary electrophoresis, and the map was similar to that of Figures 7 and 8.
- the final purified fraction was subjected to RP-HPLC and CE detection, all of which were single peaks.
- the molecular weight of the immunomodulatory active polypeptide was determined to be 2133.52 Da by MALDI-TOF-MS.
- the isoelectric point of the immunomodulatory active polypeptide was determined by CIEF to be 3.82.
- the sequence of the immunomodulatory active polypeptide was determined by a Type 491 protein sequencer: Tyr-X-Phe-Leu-Gly-Leu-Pro-Gly-X-Thr (X-amino acid).
- the liquid B used in the reversed-phase high performance liquid chromatography was 40% acetonitrile containing 0.05% trifluoroacetic acid; the other operation was the same as in Example 1.
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US11/911,201 US20080319163A1 (en) | 2005-04-21 | 2006-02-14 | Method for Isolating and Purifying Immuno-Modulating Polypeptide from Cow Placenta |
JP2008506903A JP2008536878A (ja) | 2005-04-21 | 2006-02-14 | ウシ胎盤から免疫調節ポリペプチドを分離し精製するための方法 |
EP06705640A EP1876184A1 (en) | 2005-04-21 | 2006-02-14 | A method for isolating and purifying immuno-modulating polypeptide from cow placenta |
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CNB2005100124630A CN1298736C (zh) | 2005-04-21 | 2005-04-21 | 从牛胎盘分离纯化免疫调节活性多肽的方法 |
CN200510012463.0 | 2005-04-21 |
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CN101983967A (zh) * | 2010-11-12 | 2011-03-09 | 沈阳农业大学 | 鹿胎盘寡肽的制备方法 |
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Also Published As
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US20080319163A1 (en) | 2008-12-25 |
JP2008536878A (ja) | 2008-09-11 |
CN1687107A (zh) | 2005-10-26 |
CN1298736C (zh) | 2007-02-07 |
EP1876184A1 (en) | 2008-01-09 |
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