Method for separating and purifying polypeptide for preventing cardiovascular and cerebrovascular diseases

Abstract

The invention provides a separation and purification method of polypeptide for preventing cardiovascular and cerebrovascular diseases, and relates to the technical field of prevention of cardiovascular and cerebrovascular diseases, the separation and purification method comprises a microbial fermentation link, crude separation, purification and refinement, and purity identification and quality control, the microbial fermentation link comprises strain screening and transformation, fermentation condition optimization and fermentation product harvesting, the crude separation comprises a precipitation method and an extraction method, the purification and refining comprises chromatographic separation and an electrophoresis technology, high performance liquid chromatography is adopted for purity identification and quality control, the fermentation condition optimization comprises culture medium optimization and fermentation process control, and the fermentation product harvesting comprises intracellular expression of polypeptide and extracellular secretion of polypeptide. The method has the advantages that separation is carried out step by step according to molecular weight and charge characteristics, chromatographic methods complement each other in advantages, the resolution ratio is high and mild, and the polypeptide purity can be remarkably improved. The polypeptide extracted by the purification method can be used as a raw material for preventing or treating cardiovascular and cerebrovascular diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of prevention of cardiovascular and cerebrovascular diseases, and particularly to a method for separating and purifying polypeptides for preventing cardiovascular and cerebrovascular diseases. Background Art

[0002] The method for separating and purifying polypeptides for preventing cardiovascular and cerebrovascular diseases is a systematic and scientific technical process, aiming to efficiently and accurately obtain high-purity polypeptides with the function of preventing cardiovascular and cerebrovascular diseases from complex starting materials.

[0003] Most of the raw materials used in the existing purification and separation methods are animal organs, etc. These need to work quickly during the extraction process because if the polypeptide substances cannot be extracted from animal organs within a short time, they will be decomposed by the enzymes in the cells, thus affecting the extraction effect. Therefore, it is necessary to control the timing well. If the timing is not well controlled, it is easy to cause waste. Summary of the Invention

[0004] The purpose of the present invention is to solve the deficiencies in the prior art, and a method for separating and purifying polypeptides for preventing cardiovascular and cerebrovascular diseases is proposed. The technical solution adopted by the present invention is as follows:

[0005] The method for separating and purifying polypeptides for preventing cardiovascular and cerebrovascular diseases includes a microbial fermentation step, rough separation, purification and refinement, and purity identification and quality control. The microbial fermentation step includes screening and modification of strains, optimization of fermentation conditions, and harvesting of fermentation products. The rough separation includes precipitation methods and extraction methods. The purification and refinement includes chromatographic separation and electrophoresis techniques. The purity identification and quality control adopt high-performance liquid chromatography.

[0006] As an improvement, the screening and modification of strains include primary screening and secondary screening.

[0007] As an improvement, the optimization of fermentation conditions includes optimization of the culture medium and control of the fermentation process.

[0008] As an improvement, the harvesting of fermentation products includes intracellularly expressed polypeptides and extracellularly secreted polypeptides.

[0009] As an improvement, the precipitation methods include salting out and organic solvent precipitation, and the extraction method adopts liquid-liquid extraction.

[0010] As an improvement, the chromatographic separation includes gel filtration chromatography, ion exchange chromatography, and reverse-phase high-performance liquid chromatography, and the electrophoresis techniques include isoelectric focusing electrophoresis and polyacrylamide gel electrophoresis.

[0011] The beneficial effects of the present invention are as follows:

[0012] The method for separating and purifying polypeptides for preventing cardiovascular and cerebrovascular diseases of the present invention separates step by step according to molecular weight and charge characteristics. The advantages of each chromatographic method are complementary, with high resolution and mildness, and can significantly improve the purity of polypeptides. It also has high stability, and the polypeptides extracted by this purification method can be used as raw materials for preventing or treating cardiovascular and cerebrovascular diseases. Detailed implementation mode

[0013] In order to make the content of the present invention easier to be clearly understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. The same components are denoted by the same reference numerals. It should be noted that the terms "front", "rear", "left", "right", "upper" and "lower" used in the following description refer to directions, and the terms "inner" and "outer" refer to the directions towards or away from the geometric center of a specific component respectively.

[0014] 1. Microbial fermentation link

[0015] Strain screening and modification

[0016] Primary screening: Obtain microbial strains that may produce polypeptides for preventing cardiovascular and cerebrovascular diseases from natural environments such as soil and water bodies or microbial strain preservation centers, and use specific screening media, such as media rich in precursors of physiologically active substances related to cardiovascular and cerebrovascular diseases. Isolate single colonies by the streak plate method or the spread plate method, conduct preliminary observations on the grown colonies, and select colonies with different characteristics such as morphology and color for the next test.

[0017] Re-screening: Inoculate the strains obtained from the primary screening into a liquid fermentation medium for shake flask fermentation. The composition of the fermentation medium needs to be optimized according to the type of microorganism, generally including carbon source, nitrogen source, inorganic salts, etc. After the fermentation is completed, detect the content of the target polypeptide in the fermentation broth by methods such as high performance liquid chromatography or enzyme linked immunosorbent assay, and select high-yield strains.

[0018] Fermentation condition optimization

[0019] Medium optimization: Use methods such as response surface experimental design to study the interactive effects of nutrients such as carbon source, nitrogen source, inorganic salts, and vitamins on polypeptide production.

[0020] Fermentation process control: When conducting scale-up fermentation in a fermenter, strictly control parameters such as temperature, pH value, and dissolved oxygen, monitor indicators such as biomass, substrate consumption, and product formation during the fermentation process, draw a fermentation curve, and adjust the fermentation parameters in a timely manner according to the curve changes.

[0021] Fermentation product harvesting

[0022] Intracellular expression of polypeptides: After fermentation, the microbial cells are collected by centrifugation, and the cells are broken by high-pressure homogenization or ultrasonic disruption to release the polypeptides in the cells. After disruption, the cell debris are removed by centrifugation again, and the supernatant is collected for subsequent separation and purification.

[0023] Extracellular secretion of polypeptides: directly centrifuge the fermentation broth to remove microbial cells and some impurities, and collect the supernatant. If the fermentation broth contains a lot of insoluble impurities, it can be pre-treated by filtering through filter paper or microfiltration membrane before centrifugation.

[0024] 2. Rough separation

[0025] Precipitation method

[0026] Salting out: Slowly add solid ammonium sulfate to the supernatant, stirring while adding to prevent local concentration from being too high. The first precipitation precipitates most of the impurities, collects the supernatant by centrifugation, then adds ammonium sulfate to the supernatant to precipitate the target polypeptide, collects the precipitate by centrifugation again, and dissolves the precipitate with a small amount of buffer.

[0027] Organic solvent precipitation: In a low temperature environment, slowly add pre-cooled ethanol or acetone to the polypeptide solution and stir evenly. After precipitation is completed, collect the precipitate by centrifugation and re-dissolve the precipitate with an appropriate amount of buffer.

[0028] Extraction method

[0029] Liquid-liquid extraction: Select appropriate organic phase and aqueous phase, place the two solutions in a separatory funnel, shake and mix thoroughly to allow the polypeptide to be fully distributed between the two phases, then let stand to separate the layers. After the layers are clearly separated, separate the organic phase, and wash the organic phase with an equal volume of aqueous phase 1-2 times to remove residual impurities. During back extraction, add an appropriate amount of aqueous phase to the organic phase, shake, let stand to separate the layers, and collect the aqueous phase. At this time, the target polypeptide is enriched in the aqueous phase.

[0030] 3. Purification and Refining

[0031] Chromatographic separation

[0032] Gel filtration chromatography: select a gel column with appropriate pore size, fully swell the gel with buffer, and then load it into the chromatographic column. The filling process should be uniform to avoid bubbles and stratification. When balancing the column, rinse it with at least 3-5 times the column volume of buffer. When eluting, keep the flow rate stable, collect the eluate at different time periods, and draw the elution curve by detecting the absorbance of the eluate at a specific wavelength.

[0033] Ion exchange chromatography: Select an ion exchange resin according to the charge properties of the polypeptide. The resin pretreatment includes alternating treatment with acid and alkali, followed by equilibration with a buffer solution. Load the resin into the chromatography column and equilibrate the column with 3 - 5 column volumes of buffer solution. During elution, first use a low - salt - concentration buffer solution to elute the unadsorbed impurities, and then gradually increase the salt concentration or change the pH value for gradient elution. Collect the elution peaks and determine the elution peak where the target polypeptide is located through detection.

[0034] Reverse - phase high - performance liquid chromatography: Select a suitable C18 silica gel column. The mobile phase is generally an acetonitrile - water system. The proportion of acetonitrile in the mobile phase linearly increases in a certain period of time. The injection volume is determined according to the instrument sensitivity and sample concentration, and the detection wavelength is selected according to the polypeptide structure.

[0035] First, perform a preliminary separation by gel filtration chromatography according to the molecular weight to remove most of the impurities with a large molecular weight difference from the target polypeptide, obtaining a relatively pure polypeptide mixture; then further separate by ion exchange chromatography according to the charge characteristics to remove the remaining impurities with different charge properties; finally, perform fine purification by reverse - phase high - performance liquid chromatography to obtain a high - purity target polypeptide. The combined use can give full play to the advantages of each method and gradually improve the polypeptide purity.

[0036] Electrophoresis techniques

[0037] Isoelectric focusing electrophoresis (IEF): Prepare a pH - gradient gel, which can be carried out by the carrier ampholyte method or the immobilized pH gradient method. In the carrier ampholyte method, the carrier ampholytes need to be mixed in a certain proportion and added to the gel solution. The immobilized pH gradient method uses a pre - prepared gel strip with a specific pH gradient. Mix the sample with the focusing buffer solution. During the focusing process, first perform pre - focusing at a low voltage, and then gradually increase the voltage to make the polypeptide focus at its isoelectric point position.

[0038] Polyacrylamide gel electrophoresis (PAGE): When preparing the gel, select an appropriate acrylamide concentration according to the polypeptide molecular weight range. Mix the sample with the loading buffer solution, and the loading buffer solution contains indicators such as bromophenol blue to facilitate observing the electrophoresis process. Adjust the loading volume according to the sample concentration and gel thickness. Set the electrophoresis voltage according to the gel length and thickness until the indicator migrates to an appropriate position. After electrophoresis, stain with silver staining. After cutting the gel strip in preparative PAGE, soak and elute the polypeptide with a suitable buffer solution.

[0039] First, perform a preliminary separation of the polypeptide according to the isoelectric point by isoelectric focusing electrophoresis, and then analyze each component separated by isoelectric focusing electrophoresis by polyacrylamide gel electrophoresis to further analyze the polypeptide characteristics from two dimensions of molecular weight and charge to obtain more detailed polypeptide information.

[0040] 4. Purity identification and quality control

[0041] High Performance Liquid Chromatography (HPLC): Use HPLC conditions similar to those during purification, but they can be appropriately optimized to improve resolution. Inject the standard and sample. The concentration of the standard should be accurately prepared, usually a high-purity polypeptide with known purity. Compare the retention times of the standard and the sample, and the deviation should be within the allowable range. Calculate the purity through the peak area. Purity = (peak area of the target polypeptide / total peak area) × 100%. If there are impurity peaks, analyze the relative content and possible sources of the impurity peaks. Impurities may come from microbial metabolites during fermentation, residual components of the culture medium, etc.

[0042] In the microbial fermentation stage, a wide range of strain sources combined with multi-step screening methods greatly increase the probability of screening high-yield strains. Response surface experimental design is used to optimize the culture medium, and fermentation parameters are precisely controlled. Flexible harvesting strategies for different polypeptide expression sites ensure efficient and stable fermentation, providing sufficient and high-quality raw materials for subsequent work.

[0043] In the rough separation stage, the precipitation method is simple to operate. Salting-out fractional precipitation and low-temperature organic solvent precipitation can effectively enrich polypeptides with controllable costs. The good selectivity of liquid-liquid extraction can accurately remove impurities, achieving preliminary separation and enrichment of polypeptides.

[0044] In the purification and refinement stage, chromatographic separation is carried out step by step according to molecular weight and charge characteristics. The advantages of each chromatographic method complement each other, with high resolution and mildness, which can significantly improve the purity of polypeptides. Electrophoresis technology analyzes polypeptides from multiple dimensions such as isoelectric point, molecular weight, and charge, providing rich information to help deeply understand the characteristics of polypeptides.

[0045] In the purity identification and quality control stage, the optimized HPLC detection is accurate and reliable, which can accurately determine the purity and analyze impurities, effectively guaranteeing the product quality. Generally speaking, all links of this scheme are closely coordinated, laying a solid foundation for obtaining high-purity and high-quality polypeptides for preventing cardiovascular and cerebrovascular diseases.

[0046] Separation is carried out step by step according to molecular weight and charge characteristics. The advantages of each chromatographic method complement each other, with high resolution and mildness, which can significantly improve the purity of polypeptides. And it has high stability. The polypeptides extracted by this purification method can be used as raw materials for preventing or treating cardiovascular and cerebrovascular diseases.

[0047] The above are only the preferred embodiments of this invention patent and are not intended to limit this invention patent. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of this invention patent shall be included within the protection scope of this invention patent.

Claims

1. A method for separating and purifying a polypeptide for preventing cardiovascular and cerebrovascular diseases, characterized in that: It includes microbial fermentation, crude separation, purification and refining, as well as purity identification and quality control. The microbial fermentation process includes the screening and transformation of strains, optimization of fermentation conditions and harvesting of fermentation products. Crude separation includes precipitation and extraction methods. Purification and refining includes chromatographic separation and electrophoresis technology. Purity identification and quality control use high performance liquid chromatography.

2. The method for separating and purifying a polypeptide for preventing cardiovascular and cerebrovascular diseases according to claim 1, characterized in that: The strain screening and transformation includes primary screening and secondary screening.

3. The method for separating and purifying a polypeptide for preventing cardiovascular and cerebrovascular diseases according to claim 1, characterized in that: The fermentation condition optimization includes culture medium optimization and fermentation process control.

4. The method for separating and purifying a polypeptide for preventing cardiovascular and cerebrovascular diseases according to claim 1, characterized in that: The fermentation product harvest includes polypeptides expressed within cells and polypeptides secreted outside cells.

5. The method for separating and purifying a polypeptide for preventing cardiovascular and cerebrovascular diseases according to claim 1, characterized in that: The precipitation method includes salting out and organic solvent precipitation, and the extraction method adopts liquid-liquid extraction.

6. The method for separating and purifying a polypeptide for preventing cardiovascular and cerebrovascular diseases according to claim 1, characterized in that: The chromatographic separation includes gel filtration chromatography, ion exchange chromatography and reversed phase high performance liquid chromatography, and the electrophoresis technology includes isoelectric focusing electrophoresis and polyacrylamide gel electrophoresis.