CN112646845A - Extraction and purification method of staphylococcus aureus type 5 capsular polysaccharide - Google Patents
Extraction and purification method of staphylococcus aureus type 5 capsular polysaccharide Download PDFInfo
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- 238000000605 extraction Methods 0.000 title claims description 16
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
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- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a method for extracting and purifying staphylococcus aureus type 5 capsular polysaccharide. According to the invention, firstly, staphylococcus aureus is subjected to phenolic acid treatment, then bacteria are cracked by a high-speed bead milling method, the release of capsular polysaccharide is promoted, then quaternary ammonium cationic surfactant is used for purification, and finally refined capsular polysaccharide is obtained through further purification by a cellulose gel chromatographic column, so that pollution and harm to human bodies caused by phenol, chloroform, acetone and the like can be reduced, the method is safe and environment-friendly, and the polysaccharide prepared by the method has high content, high yield and high purity, has low protein and nucleic acid content, can be directly used as an antigen without further modification, or is coupled with protein for immunization.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for extracting and purifying staphylococcus aureus type 5 capsular polysaccharide.
Background
Staphylococcus aureus is widely distributed in the natural world, and pathogenic staphylococcus frequently causes various suppurative diseases, such as abscess, septicemia or sepsis septicemia. Staphylococcus aureus is difficult to control because of the presence of a "protective film", i.e., a capsule, on the surface of the bacteria. Research shows that the capsule exists on the surface of almost all staphylococcus aureus strains, can protect bacteria from being invaded by bacteriostatic or bactericidal substances and phagocytosis of host phagocytes, is also helpful for the bacteria to adhere to the surface of the host cells to induce infection, and is an important surface structure for the bacteria to live. The capsule contains polysaccharide as main component, is one of the surface antigens with least change of bacterial structure, and has better immunogenicity. 94-100% of staphylococcus aureus is reported to contain capsular membranes, so extraction of staphylococcus aureus capsular polysaccharide has been of great interest as a target antigen of its vaccine. Currently, there are 11 capsular polysaccharide serotypes of staphylococcus aureus that have been identified, with 70-80% of staphylococcus aureus producing type 5 polysaccharide.
Extraction and purification of capsular polysaccharide are one of the key steps in the development of both capsular polysaccharide vaccines and polysaccharide protein conjugate vaccines, and the preparation process of bacterial capsular polysaccharide vaccines needs to remove impurity proteins and endotoxins as much as possible so as to reduce the side reactions of the vaccines. At present, there are many reports on extraction of staphylococcus aureus capsular polysaccharide, but the extraction methods are different. The Poplar Zhengtao and the like adopt a solid culture medium to culture the capsular polysaccharide of the type 5 Staphylococcus aureus, and thallus is collected to release the capsular polysaccharide through high-pressure cracking, but the solid culture method is not beneficial to the expanded production. The deproteinization rate is high but the polysaccharide loss is serious by using acid treatment; removing foreign proteins by using a phenol extraction method, and removing endotoxin by using ultracentrifugation, but phenol has strong toxicity and strong corrosivity and causes harm to the health of workers; phenol also has a serious environmental hazard; column chromatography is used for removing foreign proteins to purify capsular polysaccharide, but the method has small treatment capacity and high cost, different required purification methods of different bacterial capsular polysaccharide are different, and no enterprise is used for vaccine production.
The cationic surfactant has the property of precipitating nucleic acids and acidic polysaccharides from solutions of low ionic strength, under which conditions proteins and neutral polysaccharides remain in solution and form complexes with proteins and most of the polysaccharides other than acidic polysaccharides in solutions of high ionic strength, for the purpose of isolation and purification; and the method has small harm to human bodies, is easy to degrade and has great prospect in separation and purification of polysaccharide. However, the polysaccharide prepared by a single purification method has low purity and low yield. Therefore, it is necessary to develop a safe and environment-friendly method for extracting and purifying bacterial capsular polysaccharide with high yield and high purity.
Disclosure of Invention
The invention aims to provide an extraction and purification method of staphylococcus aureus type 5 capsular polysaccharide aiming at the defects of the prior art, the method utilizes quaternary ammonium salt cationic surfactant for purification, can reduce pollution and harm to human bodies caused by phenol, chloroform, acetone and the like, is safe and environment-friendly, and the polysaccharide prepared by the method has high content, high yield and high purity, has low protein and nucleic acid content, can be directly used as antigen without further modification, or can be coupled with protein for immunization.
In order to achieve the above object, the present invention adopts the following technical solutions.
A method for extracting and purifying staphylococcus aureus type 5 capsular polysaccharide, which comprises the following steps:
1) and (3) fermentation culture of staphylococcus aureus:
adding 1.8-2.2% of NaCl into a Columbia blood agar basic culture medium, carrying out autoclaving at 121 ℃ for 10-20 min, adjusting the temperature to 40-50 ℃, and adding 8-12% of newborn bovine serum to prepare an agar culture plate; inoculating a staphylococcus aureus strain seed solution identified as type 5 capsular polysaccharide to a culture medium by using a thoroughly burned inoculating loop near the flame of an alcohol burner according to the inoculation amount of 0.8-1.2 vol%, and inoculating 3-5% of CO at the temperature of 35-40 DEG C2The bacteria are cultured in the biochemical incubator for not less than 24 hours;
2) crude extraction of staphylococcus aureus capsular polysaccharide:
adding 5-10 times volume of PBS (pH 7.2-7.5) subjected to autoclaving into a culture medium to prepare a bacterial suspension, suspending the bacterial suspension in distilled water at a concentration of 1.8-2.5 mg/mL, adding 0.5-1.2% phenolic acid, treating at 80-120 ℃ for 1-1.5 h, and performing high-speed bead grinding on the bacterial suspension after phenolic acid treatment for 2-5 min; then filtering with 0.2 μm fiber membrane to remove thallus impurities, and collecting filtrate for use;
3) and (3) purifying the capsular polysaccharide:
adding a quaternary ammonium salt cationic surfactant into the filtrate prepared in the step 2), stirring while adding, standing overnight at 0-4 ℃ to precipitate polysaccharide, and dissociating the precipitate by using 1-1.5 mol/L NaCl solution; adding ethanol with the final concentration of 70-95 vol%, collecting the precipitate, and redissolving with deionized water; and separating the complex solution by cellulose column chromatography, taking a mixture of ethanol, acetic acid and water with a mass ratio of 7-9: 2-2.5: 1 as an eluent, carrying out online detection by using a differential detector at a flow rate of 0.8-1.4 mL/min, collecting according to elution peaks, respectively carrying out reduced pressure concentration and mixing to obtain a white powdery substance, namely the refined capsular polysaccharide.
The invention relates to a method for extracting and purifying staphylococcus aureus type 5 capsular polysaccharide, which comprises the steps of firstly synthesizing a culture medium to culture staphylococcus aureus, then cracking bacteria by using a high-speed bead milling method after phenolic acid treatment, promoting the release of capsular polysaccharide, then removing and purifying by using a quaternary ammonium salt cationic surfactant, and finally further purifying by using a cellulose gel chromatographic column to obtain refined capsular polysaccharide, wherein the method is safe and environment-friendly; wherein, phenolic acid treatment can inactivate bacteria and promote the release of capsular polysaccharide; bacteria can be cracked in the high-speed bead milling process, and the release of polysaccharide is further promoted; the quaternary ammonium salt cationic surfactant is added to replace organic reagents such as phenol, chloroform, acetone and the like, so that the quaternary ammonium salt cationic surfactant has little harm to human bodies and is easy to degrade; the cellulose contains cationic fibers which are all positively charged, the counter ions of the cellulose are anions and can exchange with polysaccharide anions, the polysaccharide anions are negatively charged, the exchange agent has exchange and adsorption capacity to inorganic salt ions and colloidal ions, and when the inorganic salt ions and the colloidal ions exist in the same chromatography process, competitive exchange and adsorption can be generated, so that the purposes of purification and concentration are achieved.
Further, the PBS buffer in step 2) is prepared by the following method:
taking 1.3-1.4 g of monopotassium phosphate, adding 75-80 mL of 0.1mol/L NaOH solution, and adding deionized water to dilute to 200mL to obtain the potassium phosphate-containing aqueous solution.
Further, the phenolic acid in the step 2) is p-hydroxybenzoic acid, protocatechuic acid or gallic acid.
Further, the high-speed bead grinding speed in the step 2) is 3200-3600 r/min.
Further, when the step 2) is carried out high-speed bead milling, 0.3-0.5 g of grinding balls are added into each 1mL of bacterial suspension.
Further, the grinding balls in the step 2) high-speed bead grinding process are mixed in three different sizes, wherein the sizes are 0.1mm, 0.2mm and 0.4mm respectively, and the weight ratio of the grinding balls in the three sizes is 10: 5-8: 2-5.
Further, the stirring speed in the step 3) is 150-300 r/min.
Further, the cellulose column packing in the step 3) is glucan cellulose or polydextrose cellulose.
Further, the eluent in the step 3) also contains 0.5 to 1 percent of cetylpyridinium ammonium chloride. The cetyl pyridinium chloride is a quaternary ammonium salt cationic surfactant, and the addition of the cetyl pyridinium chloride not only can thoroughly remove protein and nucleic acid, but also can improve the chromatographic separation efficiency of the cellulose column.
Further, the adding amount of the quaternary ammonium salt cationic surfactant in the step 3) is 0.5-1.4% of the weight of the filtrate.
Further, the quaternary ammonium salt cationic surfactant is (dodecyl dimethyl-2-hydroxypropyl) -ammonium acetate chloride or (dodecyl dimethyl-2-hydroxypropyl) -ammonium salicylate chloride. The quaternary ammonium salt cationic surfactant can precipitate acidic polysaccharide to achieve the purpose of purification, further improve the purity of the product capsular polysaccharide, can be directly used as an antigen without further modification, or can be coupled with protein for immunization, and obviously expand the applicable field and the availability degree of the product capsular polysaccharide.
Further, the quaternary ammonium salt cationic surfactant is prepared by the following method:
mixing carboxylic acid, dodecyl dimethyl tertiary amine and halogenated hydrocarbon in a three-necked bottle, heating to 60-80 ℃ while stirring, and reacting for 8-12 h; after the reaction is finished, dissolving the product by using sufficient acetone, cooling to form a paste, and separating two phases; removing the solvent from the product, and drying in a vacuum drying oven at 50-60 ℃ for 10-24 hours to obtain the product. The antibacterial agent is prepared by using carboxylic acid, halogenated hydrocarbon and tertiary amine for preparation reaction, the preparation method is simple, the yield is high, acetic acid or salicylic acid has a strong sterilization effect, and the sterilization effect is further improved by adding quaternary ammonium salt cations; the quaternary ammonium salt cationic surfactant can precipitate acidic polysaccharide so as to achieve the purpose of purification.
Furthermore, in the preparation step of the quaternary ammonium salt cationic surfactant, the addition molar ratio of the carboxylic acid, the dodecyl dimethyl tertiary amine and the halogenated hydrogen is 1: 1.5-3.5: 2-2.5.
Furthermore, in the preparation step of the quaternary ammonium salt cationic surfactant, the halogenated hydrogen is chloropropylene oxide, bromopropylene oxide or iodopropylene oxide.
Further, the quaternary ammonium salt cationic surfactant has a structural formula shown in formula (1) or (2):
wherein, X is Cl, Br or I.
The invention also provides staphylococcus aureus type 5 capsular polysaccharide, and the staphylococcus aureus type 5 capsular polysaccharide is prepared by the method.
The invention also provides application of the staphylococcus aureus type 5 capsular polysaccharide in immunization.
Further, the use includes the use of staphylococcus aureus type 5 capsular polysaccharide directly as an antigen without further modification, or coupled to a protein for immunization.
According to the invention, after phenolic acid treatment, bacteria are cracked by using a high-speed bead milling method, capsular polysaccharide release is promoted, quaternary ammonium salt cationic surfactant is used for purification, and finally refined capsular polysaccharide is obtained through further purification by using a cellulose gel chromatographic column, so that the method has the following beneficial effects:
1) the method utilizes the phenolic acid to treat and then utilizes the high-speed bead milling method to crack bacteria, thereby not only inactivating the bacteria and promoting the release of polysaccharide, but also reducing the content of teichoic acid in the product, and utilizing the phenolic acid to treat so as to reduce the loss of the polysaccharide caused by acetic acid treatment and improve the yield of the polysaccharide;
2) the quaternary ammonium salt cationic surfactant is used for purification, so that pollution and harm to human bodies caused by phenol, chloroform, acetone and the like can be reduced, and the method is safe and environment-friendly;
3) further purifying by using a cellulose gel chromatographic column, taking sodium acetate and water as eluent, and adding ammonium cetylpyridinium chloride to promote the extraction of polysaccharide so that the obtained polysaccharide has higher purity;
4) the polysaccharide capsule prepared by the method has high polysaccharide content and low protein and nucleic acid impurity content, and can be directly used as an antigen without further modification or coupled with protein for immunization.
The invention adopts the technical scheme to provide the model essay, makes up the defects of the prior art, and has reasonable design and convenient operation.
Drawings
In order to make the aforementioned and other objects, features, and advantages of the invention, as well as others which will become apparent, reference is made to the following description taken in conjunction with the accompanying drawings in which:
FIG. 1 is a nuclear magnetic hydrogen spectrum of (dodecyl dimethyl-2-hydroxypropyl) -ammonium chloride acetate prepared in example 1 of the present invention;
FIG. 2 is a schematic diagram showing the determination result of polysaccharide content in the crude extract of capsular polysaccharide.
Detailed Description
Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The materials, methods, and examples described herein are illustrative only and are not intended to be limiting. All publications, patent applications, patents, provisional applications, database entries, and other references mentioned herein, and the like, are incorporated by reference herein in their entirety. In case of conflict, the present specification, including definitions, will control.
The following describes the technical solution of the present invention in further detail with reference to the detailed description and the accompanying drawings.
Example 1: staphylococcus aureus type 5 capsular polysaccharide
The embodiment provides an extraction and purification method of staphylococcus aureus type 5 capsular polysaccharide, which comprises the following steps:
1) fermentation culture of staphylococcus aureus
Adding 2% NaCl into Columbia blood agar base culture medium, autoclaving at 121 deg.C for 15min, cooling to 50 deg.C in water bath, and adding 10% newborn calf serum to make agar culture plate; inoculating Staphylococcus aureus seed solution identified as type 5 capsular polysaccharide with 1.2 vol% inoculating loop near alcohol burner flame, inoculating at 37 deg.C with 5% CO2The biochemical incubator is used for culturing bacteria for 24 hours;
2) crude extraction of Staphylococcus aureus capsular polysaccharide
Taking potassium dihydrogen phosphate and 1.36g, adding 79mL of 0.1mol/L NaOH solution, adding deionized water to dilute to 200mL, and preparing PBS buffer solution with pH value of 7.4; adding 8 times volume of autoclaved PBS buffer solution into the culture medium to prepare bacterial suspension, suspending the bacterial suspension in distilled water at 2.2mg/ml, adding 0.8% p-hydroxybenzoic acid, and treating for 1h at 100 ℃; carrying out high-speed bead milling on the bacterial suspension after the p-hydroxybenzoic acid treatment for 3min at the rotating speed of 3400r/min, adding 0.4g of grinding balls into 1mL of bacterial suspension with the addition of the bacterial suspension and the grinding balls, and mixing the grinding balls with the weight ratio of 10:7:3 and the sizes of 0.1mm, 0.2mm and 0.4 mm; then filtering with 0.2 μm fiber membrane to remove thallus impurities, and collecting filtrate for use;
3) preparation of (dodecyl dimethyl-2-hydroxypropyl) -acetic acid ammonium chloride
In a three-necked bottle, 12g of acetic acid, 85.2g of dodecyl dimethyl tertiary amine and 36.8g of epoxy chloropropane are mixed, and the mixture is slowly heated to 80 ℃ while being stirred to react for 8 hours; after the reaction is finished, dissolving the product by using acetone, cooling to form a paste, and separating two phases; removing the solvent of the product, and drying in a vacuum drying oven at 50 ℃ for 24h to obtain the product.
4) Purification of capsular polysaccharides
Adding (dodecyl dimethyl-2-hydroxypropyl) -ammonium chloride acetate with the weight of 1% of the filtrate into the filtrate prepared in the step 2), stirring while adding, wherein the stirring speed is 200r/min, standing overnight at 4 ℃ for precipitating polysaccharide, and dissociating the precipitate by using 1.2mol/L NaCl solution; adding ethanol with the final concentration of 80 vol% for precipitation, collecting the precipitate, and redissolving the precipitate by using deionized water; and separating the complex solution by using a cellulose column chromatography, taking a mixture of ethanol, acetic acid, water and cetylpyridinium ammonium chloride with a mass ratio of 8.5:2:1:0.03 as an eluent, wherein the flow rate is 1.2mL/min, performing online detection by using a differential detector, collecting according to elution peaks, respectively performing reduced pressure concentration and mixing to obtain a white powdery substance, namely the refined capsular polysaccharide.
Example 2: staphylococcus aureus type 5 capsular polysaccharide
Example 2 provides another staphylococcus aureus type 5 capsular polysaccharide, which is prepared substantially identically to example 1, except that in example 2), during the crude extraction of the staphylococcus aureus capsular polysaccharide of step 2), the bacterial suspension is directly lysed by a high-speed bead mill without being treated with phenolic acid.
Example 3: staphylococcus aureus type 5 capsular polysaccharide
Example 3 provides another s.aureus type 5 capsular polysaccharide prepared substantially the same as in example 1, except that in example 3, acetic acid was used instead of p-hydroxybenzoic acid to treat the bacterial suspension during step 2) crude extraction of s.aureus capsular polysaccharide.
Example 4: staphylococcus aureus type 5 capsular polysaccharide
Example 4 provides another staphylococcus aureus type 5 capsular polysaccharide, which is prepared substantially identically to example 1, except that in example 4, in the step 2) of crude extraction of the staphylococcus aureus capsular polysaccharide, high-speed bead milling is performed using only milling balls having a size of 0.1 mm.
Example 5: staphylococcus aureus type 5 capsular polysaccharide
Example 5 provides another staphylococcus aureus type 5 capsular polysaccharide prepared substantially the same as in example 1, except that in example 5, the quaternary ammonium salt cationic surfactant used is ammonium (dodecyl dimethyl-2-hydroxypropyl) -salicylate chloride.
Example 6: staphylococcus aureus type 5 capsular polysaccharide
Example 6 provides another s.aureus type 5 capsular polysaccharide prepared substantially the same as in example 1, except that in example 6 the quaternary ammonium cationic surfactant is dodecyltrimethylammonium chloride.
Example 7: staphylococcus aureus type 5 capsular polysaccharide
Example 7 provides another staphylococcus aureus type 5 capsular polysaccharide, prepared substantially identically to example 1, except that in example 7, the eluent is water during the cellulose column chromatography separation of step 4).
Example 8: staphylococcus aureus type 5 capsular polysaccharide
Example 8 provides another s.aureus type 5 capsular polysaccharide prepared substantially the same as in example 1, except that in example 8, the eluent is sodium acetate and water in a weight ratio of 2:1 during the separation process of step 4) cellulose column chromatography.
Experimental example 1: determination of polysaccharide content of crude extract of staphylococcus aureus type 5 capsular polysaccharide
According to the principle that a phenol-sulfuric acid reagent and free hexose and uronic acid in oligosaccharide and polysaccharide have color reaction, the maximum absorption is realized at the position with the wavelength of 490nm, and the absorption value and the sugar content have a linear relation, the polysaccharide content of a sample is detected;
accurately weighing 2.5mg of glucose, dissolving with triple distilled water and fixing the volume to 25 mL; respectively sucking 0.4 mL, 0.6 mL, 0.8 mL, 1.0mL, 1.2mL and 1.4mL of standard glucose solution, and adding water to supplement to 2.0 mL; then adding 1.0mL of 6% phenol solution and 5mL of concentrated sulfuric acid, standing for 10min, shaking uniformly, standing for 20min at room temperature, and measuring the light absorption value at the wavelength of 49 nm; setting 2.0mL of triple distilled water as a blank control, and measuring a standard curve;
accurately weighing 0.07mg of the capsular polysaccharide crude product and the capsular polysaccharide refined product prepared in the embodiments 1-8 respectively, adding 1mL of triple distilled water for dissolving, sucking 100 mu L of triple distilled water, and fixing the volume to 1mL by using the triple distilled water; taking 1.0mL of solution, adding water to supplement to 2.0mL, then adding 1.0mL of 6% phenol solution and 5mL of concentrated sulfuric acid, standing for 10min, shaking up, standing at room temperature for 20min, and measuring the light absorption value at the wavelength of 490 nm;
the percentage of polysaccharide in the sample was calculated by equation (1):
Y＝0.0815X+0.0129 (1)
wherein, in the formula, (1) Y is the absorbance at 490nm, and X is the percentage content of polysaccharide;
the determination results of polysaccharide content in the crude capsular polysaccharide extracts of examples 1-8 are shown in FIG. 2:
according to the invention, the content of the capsular polysaccharide crude extract obtained by grinding staphylococcus aureus by phenolic acid treatment and high-speed bead milling is low, and further purification is required. As can be seen from fig. 2, both phenolic acid treatment and high speed bead milling affect the release of capsular polysaccharide; as can be seen from examples 1-3, the p-hydroxybenzoic acid treatment is more beneficial to the release of capsular polysaccharide than acetic acid; as can be seen from example 4, the use of milling balls of different sizes in the high-speed bead milling process results in more complete milling and more favorable release of capsular polysaccharide.
Experimental example 2: determination of content of each substance in staphylococcus aureus type 5 capsular polysaccharide
The content of polysaccharide, protein, nucleic acid and teichoic acid in the refined capsular polysaccharide prepared in examples 1-8 is measured, wherein the content of polysaccharide is measured according to the procedure of experimental example 1, the content of protein and nucleic acid in the sample is directly measured by a nucleic acid protein detector, and the measurement results are shown in table 1:
table 1: content of each substance in refined capsular polysaccharide
In the staphylococcus aureus type 5 capsular polysaccharide product prepared by the method, the polysaccharide content is higher, the highest content is 83.57%, and the content of impurities such as protein, nucleic acid and the like is lower. As can be seen from the table, both phenolic acid treatment and milling ball size have a large effect on polysaccharide content, with phenolic acid treatment, milling of different sized milling balls contributing to the release of capsular polysaccharide; it can also be seen that the protein and nucleic acid impurities in the product after phenol acid treatment are reduced; the quaternary ammonium salt cationic surfactant prepared by the invention has better purification effect; in addition, the eluent has obvious influence on the removal of protein and nucleic acid in the cellulose column chromatography process, and the addition of a small amount of cetylpyridinium chloride in the eluent is more beneficial to the purification of capsular polysaccharide.
Experimental example 3: serum potency assay
Staphylococcus aureus type 5 capsular polysaccharide prepared in example 1 was activated and derivatized, then diluted to 10 with PBS buffer9cfu/m L, 100 mul/hole coated polystyrene ELISA plate, 4 degree centigrade overnight; pouring out the liquid in the holes, filling the holes (about 300 mu l) with the washing solution, standing for 3min, removing the washing solution, repeating the steps for 3 times, and finally drying the holes on the absorbent paper; sealing with 1% gelatin, and reacting at 37 deg.C for 1 hr, wherein each well is 200 μ l; then pouring off the sealing liquid; washing, adding immune serum which is gradually diluted by PBS, and reacting for 1h at 37 ℃; washing; horseradish peroxidase-labeled goat anti-rabbit Ig G: adding 100 mul of diluted horse radish peroxidase labeled goat anti-rabbit Ig G (diluted 1: 4000) into each hole, and reacting for 1h at 37 ℃; washing; adding 100 μ l of newly prepared substrate solution into each well, and developing at 37 deg.C for 10 min; adding 50 μ l of 2M H2SO4 stop solution into each well to stop the reaction; reading an OD490nm value by using a microplate reader, and judging a detection result: the positive result is judged when the OD490nm value is more than or equal to 2.1; otherwise, the result is judged to be negative.
The titer of rabbit anti-staphylococcus aureus serum was determined by indirect ELISA, and the results are shown in table 2:
TABLE 2 serum potency assay
| Dilution factor | 200 | 400 | 800 | 1600 | 3200 | 6400 | 12800 | NC |
| Physiological saline group | OUT | OUT | 2.788 | 2.503 | 2.314 | 1.796 | 1.533 | 0.274 |
As can be seen from the table, the OD value gradually decreased with the increase of the dilution factor, and the obvious positive reaction (P/N > 2) was shown at the dilution factor of 1:12800, which indicates that the experimental requirements were met.
Conventional techniques in the above embodiments are known to those skilled in the art, and therefore, detailed descriptions thereof are omitted here. While the above detailed description has shown, described, and pointed out novel features as applied to various embodiments, it will be understood that various omissions, substitutions, and changes in the form and details of the device or method illustrated may be made without departing from the spirit of the disclosure. In addition, the various features and methods described above may be used independently of one another, or may be combined in various ways. All possible combinations and sub-combinations are intended to fall within the scope of the present disclosure. Many of the embodiments described above include similar components, and thus, these similar components are interchangeable in different embodiments. Although the present invention has been disclosed in the context of certain embodiments and examples, it will be understood by those skilled in the art that the present invention extends beyond the specifically disclosed embodiments to other alternative embodiments and/or uses and obvious modifications and equivalents thereof. Accordingly, the invention is not intended to be limited by the specific disclosure of preferred embodiments herein.
Claims (10)
1. A method for extracting and purifying staphylococcus aureus type 5 capsular polysaccharide is characterized by comprising the following steps:
1) and (3) fermentation culture of staphylococcus aureus:
adding 1.8-2.2% of NaCl into a Columbia blood agar basic culture medium, adjusting the temperature to 40-50 ℃ after autoclaving, and adding 8-12% of newborn calf serum to prepare an agar culture plate; inoculating staphylococcus aureus seed liquid identified as type 5 capsular polysaccharide to a culture medium according to the inoculation amount of 0.8-1.2 vol%, and inoculating 3-5% of CO at 35-40 DEG C2The bacteria are cultured in the biochemical incubator for not less than 24 hours;
2) crude extraction of staphylococcus aureus capsular polysaccharide:
adding 5-10 times volume of PBS (pH 7.2-7.5) subjected to autoclaving into a culture medium to prepare bacterial suspension, suspending the bacterial suspension in distilled water at a concentration of 1.8-2.5 mg/mL, adding 0.5-1.2% of phenolic acid, treating for 1-1.5 h at 80-120 ℃, and performing high-speed bead grinding on the bacterial suspension after the phenolic acid is treated for 2-5 min; then filtering with 0.2 μm fiber membrane to remove thallus impurities, and collecting filtrate for use;
3) and (3) purifying the capsular polysaccharide:
adding a quaternary ammonium salt cationic surfactant into the filtrate prepared in the step 2), stirring while adding, standing overnight at 0-4 ℃ to precipitate polysaccharide, and dissociating the precipitate by using 1-1.5 mol/L NaCl solution; adding ethanol with the final concentration of 70-95 vol%, collecting the precipitate, and redissolving with deionized water; separating the complex solution by cellulose column chromatography, taking a mixture of ethanol, acetic acid and water with a mass ratio of 7-9: 2-2.5: 1 as an eluent, carrying out online detection by using a differential detector, collecting according to an elution peak, and carrying out reduced pressure concentration and mixing to obtain a white powdery substance, namely the refined capsular polysaccharide.
2. The method of claim 1, wherein the phenolic acid of step 2) is p-hydroxybenzoic acid, protocatechuic acid, gallic acid.
3. The method according to claim 1 or 2, wherein 0.3-0.5 g of grinding balls are added into 1mL of the bacterial suspension in the step 2) high-speed bead grinding process, the grinding balls are mixed in three different sizes, namely 0.1mm, 0.2mm and 0.4mm, and the weight ratio of the grinding balls in the three sizes is 10: 5-8: 2-5.
4. The method according to any one of claims 1 to 3, wherein the quaternary ammonium salt cationic surfactant in the step 3) is (dodecyl dimethyl-2-hydroxypropyl) -ammonium acetate chloride or (dodecyl dimethyl-2-hydroxypropyl) -ammonium salicylate chloride.
5. The method according to any one of claims 1 to 4, wherein the quaternary ammonium salt cationic surfactant is prepared by a method comprising:
mixing carboxylic acid, dodecyl dimethyl tertiary amine and halogenated hydrocarbon according to a molar ratio of 1: 1.5-3.5: 2-2.5, heating to 60-80 ℃ while stirring, and reacting for 8-12 h; after the reaction is finished, dissolving the product by using sufficient acetone, cooling to form a paste, and separating two phases; removing the solvent from the product, and drying the product in vacuum at 50-60 ℃ for 10-24 hours to obtain the product.
6. The process according to claim 5, wherein the halogenated hydrocarbon is epichlorohydrin, epibromohydrin or epiiodohydrin.
7. The method according to any one of claims 1 to 6, wherein the eluent in step 3) further comprises 0.5 to 1% cetylpyridinium ammonium chloride.
8. The method as claimed in any one of claims 1 to 7, wherein the cellulose column packing material in the step 3) is cellulose dextran or cellulose polydextrose.
9. Staphylococcus aureus type 5 capsular polysaccharide, prepared by the method of any one of claims 1 to 8.
10. Use of a staphylococcus aureus type 5 capsular polysaccharide according to any one of claims 1-9 in immunisation, comprising using a staphylococcus aureus type 5 capsular polysaccharide prepared by a process according to any one of claims 1-9 as an antigen directly without further modification, or coupled to a protein for immunisation.
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