CN112898443B - Method for extracting and purifying lipooligosaccharide and oligosaccharide in thallus - Google Patents

Method for extracting and purifying lipooligosaccharide and oligosaccharide in thallus Download PDF

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CN112898443B
CN112898443B CN202110119637.2A CN202110119637A CN112898443B CN 112898443 B CN112898443 B CN 112898443B CN 202110119637 A CN202110119637 A CN 202110119637A CN 112898443 B CN112898443 B CN 112898443B
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胡浩
蒋浩然
雷丹
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Suzhou Weichao Biotechnology Co ltd
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    • C07—ORGANIC CHEMISTRY
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Abstract

The invention discloses a method for extracting and purifying lipooligosaccharide in thalli, which comprises the following steps: a. collecting dry bacterial sludge; b. mixing the dry bacterial sludge with an isobutyric acid ammonia water solution, centrifuging, and collecting a supernatant, wherein the isobutyric acid ammonia water solution is a mixture of isobutyric acid and concentrated ammonia water; c. mixing the supernatant obtained in the step b with ethanol, standing, centrifuging, and collecting precipitate; d. c, extracting the precipitate obtained in the step c by using a chloroform methanol solution, and collecting an organic phase; e. drying the organic phase obtained in step d. The invention also discloses a method for extracting and purifying oligosaccharide in thallus, which comprises the steps of hydrolyzing and purifying the product obtained in the step e.

Description

Method for extracting and purifying lipooligosaccharide and oligosaccharide in thallus
Technical Field
The invention relates to the technical field of oligosaccharide extraction and purification, in particular to a method for extracting and purifying lipooligosaccharide and oligosaccharide in thalli.
Background
Pertussis (pertussis) is an acute respiratory infectious disease caused by Bordetella pertussis (Bordetella pertussis, abbreviated as Bordetella pertussis) which is the only natural host, and despite good vaccination coverage, a large number of patients remain annually and cause death in some patients. Currently, there are two pertussis vaccines, one is the whole cell pertussis vaccine (wP) and the other is the acellular pertussis vaccine (aP). aP, while providing protection against bordetella infection, does not completely prevent its transmission and is significantly less effective at preventing infection than wP. Many studies have demonstrated that the acellular pertussis vaccine in use is associated with the recurrence of pertussis disease.
Lipooligosaccharide (LOS) on the surface of bordetella pertussis is a pathogenic substance of bacteria, and the trisaccharide structure of the outermost layer is also a specific antigen of bordetella pertussis type (fig. 1). It has been demonstrated by scholars that the terminal trisaccharide structure of bordetella pertussis LOS is the target for the action of specific bactericidal antibodies. The research also preliminarily proves that the conjugate prepared by using the O-SP-ABS can enable the mice to generate functional antibodies with certain bactericidal effect. Therefore, the pertussis oligosaccharide can provide a new idea for developing a novel pertussis antigen component. LOS is extracted by a traditional hot phenol water method, the extraction period is long, the toxic solvent phenol is used to cause harm to operators, the risk of toxic substances exists in final products, and the process for removing residual toxic substances in subsequent products is complex.
Disclosure of Invention
Therefore, it is necessary to provide a method for extracting and purifying lipooligosaccharide and oligosaccharide in the bacterial cells, aiming at the problems of the traditional hot phenol water extraction method.
A method for extracting and purifying lipooligosaccharide in thalli comprises the following steps:
a. collecting dry bacterial sludge;
b. mixing the dry bacterial sludge with an isobutyric acid ammonia water solution, centrifuging, and collecting a supernatant, wherein the isobutyric acid ammonia water solution is a mixture of isobutyric acid and concentrated ammonia water;
c. mixing the supernatant obtained in the step b with ethanol, standing, centrifuging, and collecting precipitate;
d. c, extracting the precipitate obtained in the step c by using a chloroform methanol solution, and collecting an organic phase;
e. and d, drying the organic phase obtained in the step d.
In some embodiments, the mass concentration of the concentrated ammonia water is 22-28%.
In some of these embodiments, the volume ratio of the isobutyric acid to the concentrated aqueous ammonia in the isobutyric acid aqueous ammonia solution is (4-6): 3.
In some embodiments, in step b, 8ml to 12ml of the isobutyric acid ammonia solution is added per gram of the dry bacterial sludge.
In some of these embodiments, the concentration of ethanol in the mixture of supernatant and ethanol in step c is (75-85) ml/100ml.
In some of these embodiments, the chloroform to methanol volume ratio of the chloroform methanol solution is (1-2.5): 1.
In some of these embodiments, step d is preceded by a MgSO-containing composition 4 Resuspending the pellet from step c in the buffer of (1), and extracting the resuspension solution with chloroform-methanol solution.
In some of these embodiments, the MgSO-containing material 4 pH of the buffer solution of (3) is 7.4 to 7.8, mgSO 4 The concentration is 1.5 mmol/L-2.5 mmol/L; preferably, the content of MgSO is one gram of dried bacterial sludge 4 The amount of the buffer solution (2) used is 5 to 10ml.
In some of these embodiments, the chloroform methanol solution used in step d is contacted with the MgSO-containing solution 4 The volume ratio of the buffer solution (1) to (4) is 1.
In some of these embodiments, the organic phase is subjected to rotary evaporation drying in step e; preferably, the rotary evaporation drying process comprises the steps of adding water for redissolving, dialyzing and vacuum freeze drying the dried substance in sequence.
In some of these embodiments, step a comprises:
a1, centrifuging fermentation liquor and collecting thalli;
a2, uniformly mixing purified water and thalli, and centrifugally collecting bacterial sludge;
a3, mixing the bacterial sludge obtained in the step a2 with ethanol, and centrifuging to collect bacterial sludge;
and a4, mixing the bacterial sludge obtained in the step a3 with methanol, and centrifuging to collect dry bacterial sludge.
In some of these embodiments, the bacteria are selected from any one or more of bordetella pertussis, escherichia coli, bordetella parapertussis, salmonella, shigella, pseudomonas, neisseria, haemophilus.
The method for extracting and purifying the oligosaccharides in the thalli is to obtain the lipooligosaccharide according to the method for extracting and purifying the lipooligosaccharide in the thalli and then hydrolyze the lipooligosaccharide.
The invention provides a method for extracting lipooligosaccharide and oligosaccharide from thalli. The LOS is then hydrolyzed to remove lipids and obtain Oligosaccharides (OS). Compared with the traditional hot phenol water method for extracting LOS, the method has the advantages that the extraction period is short, toxic solvent phenol is not added, the harm to operators is avoided, the risk of toxic substances in the final product is avoided, the complicated process steps of removing the residual toxic substances in the subsequent products are avoided, and the cost is reduced. In addition, the inventor finds that the purity of LOS can be obviously improved by improving the concentration of ammonia water in the extracting solution, and the subsequent purification difficulty is reduced; the purification process adopts an ethanol precipitation method to precipitate LOS, further purify the LOS, and the precipitate is extracted with chloroform-methanol to further remove impurities such as nucleic acid protein, and finally obtain pure LOS. Hydrolysis of LOS yields substantially pure OS.
Drawings
FIG. 1 is a Bordetella pertussis LOS structure;
FIG. 2 is a schematic diagram of a LOS extraction and purification process according to an embodiment of the present invention;
FIG. 3 is a schematic diagram of an OS extraction and purification process according to an embodiment of the present invention;
FIG. 4 is a LOS electrophoretogram extracted with concentrated ammonia water and 1M ammonia water according to an embodiment and a comparative example of the present invention;
FIG. 5 is an LOS electrophoretogram of ammonia isobutyrate extracted with different ratios according to one embodiment of the present invention and a comparative example;
FIG. 6 is an LOS electrophoretogram obtained by chloroform-methanol extraction at different ratios according to an embodiment of the present invention and a comparative example;
FIG. 7A is a SDS-PAGE image of a sample of an LOS extraction process according to one embodiment of the invention;
FIG. 7B is an SDS-PAGE image of a purified LOS sample according to one embodiment of the invention;
FIG. 8A is a schematic representation of a purified LOS sample in a countercurrent immunoelectrophoresis format according to one embodiment of the present invention;
FIG. 8B is a diagram of a purified OS sample countercurrent immunoelectrophoresis in accordance with one embodiment of the present invention;
FIG. 9 is an OS 600MHz 1H NMR spectrum.
Detailed Description
To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all 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 terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
The embodiment of the invention provides a method for extracting and purifying lipooligosaccharide in thallus, which comprises the following steps:
a. collecting dry bacterial sludge;
b. mixing the dry bacterial sludge with an isobutyric acid ammonia water solution, centrifuging, and collecting a supernatant, wherein the isobutyric acid ammonia water solution is a mixture of isobutyric acid and concentrated ammonia water;
c. mixing the supernatant obtained in the step b with ethanol, standing, centrifuging, and collecting precipitate;
d. c, extracting the precipitate obtained in the step c by using a chloroform methanol solution, and collecting an organic phase;
e. drying the organic phase obtained in step d.
The invention provides a method for extracting lipooligosaccharide and oligosaccharide from thalli, wherein an isobutyric acid ammonia water solution is added into dry bacteria for extraction, and pure LOS is obtained by ethanol precipitation and chloroform-methanol solution extraction. The LOS is then hydrolyzed to remove lipids and obtain Oligosaccharides (OS). Compared with the traditional hot phenol water method for extracting LOS, the method has the advantages that the extraction period is short, toxic solvent phenol is not added, the harm to operators is avoided, the risk of toxic substances in the final product is avoided, the complicated process steps of removing the residual toxic substances in the subsequent products are avoided, and the cost is reduced. In addition, the inventor finds that the purity of LOS can be obviously improved by improving the concentration of ammonia water in the extracting solution, and the subsequent purification difficulty is reduced; the purification process adopts an ethanol precipitation method to precipitate LOS, further purify the LOS, and the precipitate is extracted with chloroform-methanol to further remove impurities such as nucleic acid protein, and finally obtain pure LOS. LOS is hydrolyzed to obtain pure OS.
In some embodiments, step a comprises:
a1, centrifuging fermentation liquor and collecting thalli;
a2, uniformly mixing purified water and thalli, and centrifugally collecting bacterial sludge;
a3, mixing the bacterial sludge obtained in the step a2 with ethanol, and centrifuging to collect bacterial sludge;
and a4, mixing the bacterial sludge obtained in the step a3 with methanol, and centrifuging to collect dry bacterial sludge.
The thallus extracted from the fermentation liquor is washed by purified water, ethanol and methanol, so that other cell components (such as phospholipid, fatty acid and the like) which are possibly extracted together with LOS can be removed while the bacteria are dehydrated, and the subsequent purification work of the LOS is simplified.
In some embodiments, in step a2, the ratio of the cells to the purified water is 1. Preferably 1.
In some embodiments, it is preferable to use an ethanol solution with a volume fraction of 80% to 90% in step a 3. The adding ratio of the bacterial sludge to the ethanol solution can be 1-1 (w/v). Preferably 1.
In some embodiments, in step a3, the mixing of the bacterial sludge and the ethanol is performed by heating, stirring and mixing at 45-55 ℃. The heating and stirring time is preferably 10min.
In some embodiments, in step a4, the ratio of the bacterial sludge to methanol is 1 to 1 (w/v). Preferably 1.
In some embodiments, in step a, the purified water, ethanol and methanol are recycled to treat the bacterial cells, wherein the recycling times are 2 times and more than 2 times.
In some embodiments, in step a, each solvent is added in a proportion corresponding to the weight of wet bacterial sludge.
In some embodiments, the volume ratio of the isobutyric acid to the concentrated aqueous ammonia in the isobutyric acid aqueous ammonia solution is (4-6): 3, preferably (4.5-5.5): 3.
In some embodiments, the concentrated aqueous ammonia in the aqueous ammonia isobutyrate solution in step b refers to an aqueous ammonia having a mass concentration of ammonia of 22% to 28%. Specifically, the mass concentration of the concentrated ammonia water may be 22%, 23%, 24%, 25%, 26%, 27%, 28%. Preferably 25% to 28%, and the concentration of ammonia water is increased, within which the purity and extraction rate of LOS obtained by extraction can be further increased.
In some embodiments, in step b, 8ml to 12ml of the aqueous ammonia isobutyrate solution is added per gram of the dried bacterial sludge. Specifically, the concentration may be 8ml, 9ml, 10ml, 11ml or 12ml.
In some embodiments, the ethanol employed in step c is absolute ethanol.
In some embodiments, the concentration of ethanol in the mixture of the supernatant and ethanol in step c may be (75-85) ml/100ml. Specifically, the concentration may be 75ml/100ml, 78ml/100ml, 80ml/100ml, 82ml/100ml, 85ml/100ml, or the like.
In some embodiments, the chloroform-methanol solution is a mixture of chloroform and methanol.
In some embodiments, the chloroform to methanol volume ratio in the chloroform-methanol solution may be (1-2.5): 1. Specifically, the following ratio can be 1.
In some embodiments, step d is preceded by a MgSO-containing composition 4 Resuspending the pellet from step c in the buffer of (1), and extracting the resuspension solution with chloroform-methanol solution.
In some embodiments, the MgSO containing 4 The pH of the buffer solution (2) is 7.4 to 7.8. Containing MgSO 4 In the buffer of (2), mgSO 4 The concentration can be 1.5 mmol/L-2.5 mmol/L, and the Tris concentration can be 45 mmol/L-55 mmol/L. Preferably, the dried bacterial sludge contains MgSO per gram 4 The amount of the buffer solution (2) used is 5 to 10ml.
In some embodiments, the chloroform methanol solution used in step d is contacted with the MgSO-containing solution 4 The volume ratio of the buffer solution (1) to (4) is 1.
Preferably, step d further comprises: and c, extracting the precipitate obtained in the step c by using a chloroform methanol solution, collecting an intermediate phase between the organic phase and the water phase, repeatedly extracting the intermediate phase by using the chloroform methanol solution, and collecting the organic phase. The inventors have found that, in fact, the intermediate phase also contains some LOS which is not clearly extracted into the organic phase, and by repeating the extraction, the yield of the final extracted LOS can be improved. The number of times of repeated extraction is 1 to 5, preferably 2 to 4, and most preferably 3.
In some embodiments, the MgSO-containing material of step d 4 The buffer solution of (a) may be Tris-MgSO 4 And (4) a buffer solution.
Step d said containing MgSO 4 The buffer of (3) may be replaced with purified water.
In some embodiments, the organic phase is subjected to rotary evaporation drying in step e. Preferably, the rotary evaporation drying method comprises the steps of adding water for redissolution, dialyzing and vacuum freeze drying on the dried substance in sequence. The dialysis pore size can be 1K.
The embodiment of the invention also provides a method for extracting and purifying oligosaccharides in thallus, wherein the method for extracting and purifying the lipooligosaccharide in the thallus is used for obtaining the lipooligosaccharide, and then the lipooligosaccharide is hydrolyzed.
In some embodiments, the method of hydrolyzing lipooligosaccharides can be acid hydrolysis. The acid used for acid hydrolysis may be acetic acid. The method comprises the following specific steps: dissolving LOS into 2 mg/ml-30 mg/ml with water, adding glacial acetic acid until the final concentration of the acetic acid is 0.8% -1.5%, heating for hydrolysis, and adjusting the pH value to 6.8-7.2 to terminate the reaction; the supernatant was collected by centrifugation, filtered through a 0.45 to 0.7 μm filter, and the filtrate was lyophilized under vacuum to obtain OS. The time for the hydrolysis with acetic acid may be 1 hour to 4 hours, preferably 2 hours.
In some embodiments, further comprising a step of purifying the OS obtained by hydrolysis.
The purification method may be column chromatography. The method specifically comprises the following steps: dissolving OS in water for injection, purifying with chromatography column, and collecting eluate at buffer flow rate of 2 ml/min. Measuring chemical distribution of the eluate by phenol-sulfuric acid method, collecting the first peak of chemical distribution, mixing, dialyzing, and dialyzing with injectable water at 4-8 deg.C for 2-4 days. And (5) freeze-drying to obtain refined OS. The chromatographic column can be any one of G25 and P-4 chromatographic columns. The buffer was water for injection or a pyridine acetic acid solution (pyridine: acetic acid: water 4. The dialysis pore size may be 1K.
The thallus applicable to the extraction and purification method of the invention can be selected from any one or more of bordetella pertussis, escherichia coli, parapertussis, salmonella, shigella, pseudomonas, neisseria, haemophilus, and other components containing lipopolysaccharide or lipooligosaccharide.
The lipooligosaccharide of the invention particularly refers to lipooligosaccharide on the cell wall of thallus.
The oligosaccharide obtained by the extraction and purification method can be applied to the preparation of molecular adjuvants, pertussis vaccines, therapeutic products, animal vaccines and the like.
The following are specific examples.
Example 1
The Bordetella pertussis LOS extraction and purification method is shown in a process diagram of figure 2 and specifically comprises the following implementation steps:
(1) Collecting and washing thallus
Taking 100g of bacterial sludge centrifugally collected from pertussis fermentation liquor, adding 500ml of purified water, stirring uniformly, centrifugally collecting thalli, and repeating for 2 times; resuspending the thallus in 300ml of 86% ethanol, stirring for 10min in 52 ℃ water bath, centrifuging, discarding the supernatant, and repeating for 2 times; the cells were resuspended in 300ml of methanol, stirred and mixed well, centrifuged and the supernatant was discarded, and the procedure was repeated 2 times to obtain 45g of dry cells.
(2) LOS extraction and crude purification
Adding the dry bacteria with the volume ratio of 5: 450ml of 3-isobutyric acid ammonia solution is uniformly dispersed, stirred for 5 minutes and centrifuged to take the supernatant. The aqueous solution of isobutyric acid consists of isobutyric acid and concentrated aqueous ammonia. The concentration of the concentrated ammonia water is 24 percent.
Adding ethanol into the supernatant until the final concentration of ethanol reaches 80%, mixing, standing at 2-8 deg.C overnight, centrifuging, and collecting precipitate to obtain crude pure LOS.
(3) LOS pure
Adding Tris-MgSO into crude LOS 4 315ml of buffer (containing 2mmol/L MgSO4, 50mmol/L Tris, pH 7.6) and suspending to uniformly disperse LOS;
resuspending the crude pure LOS solution, adding a solution with the volume ratio of 2: 788ml of chloroform-methanol solution is shaken vigorously for 10 minutes and then centrifuged to separate layers, an upper layer is absorbed and discarded, and an intermediate layer and a lower layer are collected respectively; adding Tris-MgSO into the interlayer solution 4 Buffer to 315ml, add 2: 788ml of 1 chloroform-methanol solution was subjected to the second extraction, and the lower phase was collected.
Mixing the two extracted lower phases, rotary evaporating to dry to obtain LOS, adding water, resuspending, placing into 1K dialysis bag, dialyzing with purified water at 2-8 deg.C for 2 days, and changing water 2 times per day.
The dialyzed solution was lyophilized in vacuo to obtain refined LOS 330mg.
The LOS electrophorograms obtained from the extraction process samples and purification are shown in FIG. 7A and FIG. 7B, respectively.
Example 2
The method for extracting and purifying bordetella pertussis LOS comprises the following specific implementation steps:
(1) Collecting and washing thallus
Taking 240g of bacterial sludge centrifugally collected from pertussis fermentation liquor, adding 1200ml of purified water, stirring uniformly, centrifugally collecting thalli, and repeating for 2 times; resuspending the thallus in 600ml of 86% ethanol, stirring for 15min in 52 ℃ water bath, centrifuging, discarding the supernatant, and repeating for 2 times; the cells were resuspended in 480ml of methanol, stirred well, centrifuged and the supernatant discarded, and repeated 2 times to obtain 96.8g of dry cells.
(2) LOS extraction and crude purification
Adding the dry bacteria into the mixture according to the volume ratio of 5: 970ml of 3-isobutyric acid ammonia solution is evenly dispersed, stirred for 5 minutes and centrifuged to take the supernatant. The aqueous ammonia isobutyrate solution consists of isobutyric acid and concentrated aqueous ammonia. The concentration of the concentrated ammonia water is 25 percent.
Adding ethanol into the supernatant until the final concentration of ethanol reaches 80%, mixing, standing at 2-8 deg.C overnight, centrifuging, and collecting precipitate to obtain crude pure LOS.
(3) LOS pure and refined
680ml of Tris-MgSO4 buffer solution (containing 2mmol/L MgSO4 and 50mmol/L Tris, pH 7.6) is added into the crude pure LOS, and the LOS is dispersed uniformly through resuspension;
resuspending the crude pure LOS solution, adding a volume ratio of 2:1, shaking the chloroform methanol solution 1700ml vigorously for 15 minutes, centrifuging the solution to layer, absorbing an upper phase, discarding the upper phase, and collecting a middle phase and a lower phase respectively; adding Tris-MgSO into the interlayer solution 4 Buffer to 680ml, add 2: a second extraction was performed with 1700ml of 1 chloroform-methanol solution and the lower phase was collected.
Mixing the two extracted lower phases, rotary evaporating to dry to obtain LOS, adding water, resuspending, placing into 1K dialysis bag, dialyzing with purified water at 2-8 deg.C for 2 days, and changing water 3 times per day.
Vacuum freeze drying the dialyzed solution to obtain refined LOS 695.2mg.
Example 3
The Bordetella pertussis LOS extraction and purification method comprises the following specific implementation steps:
(1) Collecting and washing thallus
Taking 400g of bacterial sludge centrifugally collected from pertussis fermentation liquor, adding 2000ml of purified water, stirring uniformly, centrifugally collecting thalli, and repeating for 2 times; resuspending the thallus in 1000ml 86% ethanol, stirring in 52 deg.C water bath for 10min, centrifuging, discarding supernatant, and repeating for 2 times; the thallus is resuspended in 800ml of methanol, evenly stirred, centrifuged and the supernatant is discarded, and the process is repeated for 2 times to obtain 170g of dry thallus.
(2) LOS extraction and crude purification
Adding the dry bacteria with the volume ratio of 5: 1700ml of 3-isobutyric acid ammonia solution was uniformly dispersed, stirred for 20 minutes, and centrifuged to obtain the supernatant. The aqueous solution of isobutyric acid consists of isobutyric acid and concentrated aqueous ammonia. The concentration of the concentrated ammonia water is 25 percent.
Adding ethanol into the supernatant until the final concentration of the ethanol is 80%, mixing uniformly, standing overnight at 2-8 ℃, centrifuging and collecting precipitate to obtain crude pure LOS.
(3) LOS pure and refined
Adding Tris-MgSO4 buffer solution (containing 2mmol/L MgSO 4) into crude pure LOS 4 50mmol/L Tris, pH 7.6) 1190ml, and resuspending to make LOS disperse uniformly;
resuspending the crude pure LOS solution, adding a solution with the volume ratio of 2: 2975ml of 1 chloroform methanol solution, shaking vigorously for 20 minutes, centrifuging to separate the solution, sucking the upper phase, discarding, and collecting the middle layer and the lower phase respectively; adding Tris-MgSO to the intermediate layer solution 4 Buffer solution is added to 1190ml, and then the volume ratio is added to 2: 2975ml of chloroform-methanol solution is used for the second extraction, and the lower phase and the middle phase are respectively collected; the middle layer was extracted again and the lower phase was collected.
Mixing the lower phases, rotary evaporating to obtain LOS, adding water, resuspending, loading into 1K dialysis bag, dialyzing with purified water at 2-8 deg.C for 2 days, and changing water 3 times per day.
Vacuum freeze drying dialyzed solution to obtain refined LOS 1582.8mg.
Example 4
The OS extraction and purification method has a process diagram as shown in FIG. 3, and comprises the following specific implementation steps:
200mg of purified LOS obtained by the extraction and purification method of example 1 is weighed, 40ml of purified water is added to dissolve the LOS, 400 mu l of glacial acetic acid is added until the final concentration of acetic acid is 1%, and the hydrolysis is carried out for 2h at 100 ℃.
The supernatant was collected by centrifugation, adjusted to pH 7.0 by the addition of 0.5M sodium hydroxide, collected by centrifugation, and lyophilized to obtain crude OS.
OS was dissolved to 10mg/ml with a pyridine acetic acid solution (pyridine: acetic acid: water 4, pH 4.7), and the eluate was collected using a P-4 column chromatography with a pyridine acetic acid solution as a buffer at a flow rate of 1 ml/min. Measuring chemical distribution of the eluate by phenol-sulfuric acid method, collecting the first peak of chemical distribution, mixing, dialyzing with injectable water at 4-8 deg.C for 3 days, and changing water 2 times per day. The dialysate was lyophilized to give refined OS 36.5mg with a yield of 18.2%.
The results of the countercurrent immunoelectrophoresis of the LOS obtained in example 1 and the OS obtained in example 4 are shown in FIGS. 8A and 8B, respectively. The result shows that LOS and OS of the purified products have precipitation reaction with pertussis immune serum. The OS nmr spectrum was consistent with that of the standard substance, as shown in fig. 9.
Example 5
The OS extraction and purification method comprises the following specific implementation steps:
1500mg of purified LOS obtained by the extraction and purification method of example 1 is weighed, 300ml of purified water is added to dissolve the LOS, 3ml of glacial acetic acid is added until the final concentration of acetic acid is 1%, and hydrolysis is carried out for 2h at 100 ℃.
The supernatant was collected by centrifugation, adjusted to pH 7.19 by the addition of 0.5M sodium hydroxide, collected by centrifugation, and lyophilized to obtain crude OS.
OS was dissolved in water for injection to 10mg/ml, and the eluate was collected using a G25 column chromatography with a flow rate of 2ml/min as the buffer. Measuring chemical distribution of the eluate by phenol-sulfuric acid method, collecting the first peak of chemical distribution, mixing, dialyzing with injectable water at 4-8 deg.C for 2 days, and changing water 2 times per day. The dialysate was lyophilized to obtain 290mg of refined OS, with a yield of 19.3%.
The results of the impurity contents of purified LOS and purified OS in examples 1 to 5 are shown in Table 1. Obtain high-purity LOS and OS, wherein the content of impurity protein in the LOS is less than 5.3%, the content of nucleic acid is less than 6.7%, the content of impurity protein in refined OS is less than 2.1%, the content of nucleic acid is less than 0.7%, and the content of bacterial endotoxin is less than 3EU/mg.
TABLE 1 purified LOS and refined OS impurity levels
Sample (I) Protein content (%) Nucleic acid content (%) Endotoxin content EU/mg
Example 1 LOS-1 5.3 2.98 /
Example 2 LOS-2 4.3 6.7 /
EXAMPLE 3 LOS-3 4.5 6 /
Example 4 OS-1 2.08 0.71 0.991
Example 5 OS-2 0.8 0.3 2.999
Comparative example 1
Comparative example 1 is substantially the same as example 1 except that the aqueous ammonia in the aqueous ammonia isobutyrate solution is 1M dilute aqueous ammonia.
The method for extracting and purifying bordetella pertussis LOS comprises the following specific implementation steps:
(1) Collecting and washing thallus
Taking 100g of bacterial sludge centrifugally collected from pertussis fermentation liquor of the same batch as in example 1, adding 500ml of purified water, stirring uniformly, centrifugally collecting thalli, and repeating for 2 times; resuspending the thallus in 300ml of 86% ethanol, stirring for 10min in 52 ℃ water bath, centrifuging, discarding the supernatant, and repeating for 2 times; the cells were resuspended in 300ml of methanol, stirred and mixed well, centrifuged and the supernatant was discarded, and the procedure was repeated 2 times to obtain 45g of dry cells.
(2) LOS extraction and crude purification
Adding the dry bacteria into the mixture according to the volume ratio of 5: 450ml of 3-isobutyric acid ammonia solution is uniformly dispersed, stirred for 5 minutes and centrifuged to take the supernatant. The aqueous ammonia isobutyrate solution consists of isobutyric acid and 1M aqueous ammonia.
Adding ethanol into the supernatant until the final concentration of ethanol reaches 80%, mixing, standing at 2-8 deg.C overnight, centrifuging, and collecting precipitate to obtain crude pure LOS.
The electrophoretic contrast of the crude pure LOS extracted in example 1 and comparative example 1 is shown in fig. 4.
Comparative example 2
Comparative example 2 is substantially the same as example 1 except that the isobutyric acid-concentrated aqueous ammonia volume ratio in the isobutyric acid aqueous ammonia solution is different.
The Bordetella pertussis LOS extraction and purification method comprises the following specific implementation steps:
(1) Collecting and washing thallus
Taking 100g of bacterial sludge centrifugally collected from pertussis fermentation liquor of the same batch as in example 1, adding 500ml of purified water, stirring uniformly, centrifugally collecting thalli, and repeating for 2 times; resuspending the thallus in 300ml of 86% ethanol, stirring for 10min in 52 ℃ water bath, centrifuging, discarding the supernatant, and repeating for 2 times; the cells were resuspended in 300ml of methanol, stirred and mixed well, centrifuged and the supernatant was discarded, and the procedure was repeated 2 times to obtain 45g of dry cells.
(2) LOS extraction and crude purification
Respectively taking 9g of dry thalli, and adding 90ml of isobutyric acid ammonia water, wherein the volume ratio of isobutyric acid ammonia water is 3: 5. 4: 4. 5: 3. 6: 2. 7:1, stirring for 5 minutes after uniform dispersion, and centrifuging to take the supernatant. The aqueous ammonia isobutyrate solution consisted of isobutyric acid and 25% aqueous ammonia.
Adding ethanol into the supernatant until the final concentration of the ethanol is 80%, mixing uniformly, standing overnight at 2-8 ℃, centrifuging and collecting precipitate to obtain crude pure LOS.
The electrophoretic contrast of the crude pure LOS extracted in example 1 and comparative example 2 is shown in fig. 5.
Comparative example 3
Comparative example 3 is essentially the same as example 1 except that the chloroform to methanol volume ratio in the chloroform-methanol solution is different.
The method for extracting and purifying bordetella pertussis LOS comprises the following specific implementation steps:
(1) Collecting and washing thallus
Taking 100g of bacterial sludge centrifugally collected from pertussis fermentation liquor of the same batch as in example 1, adding 500ml of purified water, stirring uniformly, centrifugally collecting thalli, and repeating for 2 times; resuspending the thallus in 300ml of 86% ethanol, stirring for 10min in 52 ℃ water bath, centrifuging, discarding the supernatant, and repeating for 2 times; the thalli is suspended in 300ml of methanol, evenly stirred, centrifuged and supernatant is discarded, and the process is repeated for 2 times to obtain 45g of dry thalli.
(2) LOS extraction and crude purification
Adding the dry bacteria with the volume ratio of 5: 450ml of 3-isobutyric acid ammonia solution is uniformly dispersed, stirred for 5 minutes and centrifuged to take the supernatant. The aqueous ammonia isobutyrate solution consisted of isobutyric acid and 25% aqueous ammonia.
Adding ethanol into the supernatant until the final concentration of ethanol reaches 80%, mixing, standing at 2-8 deg.C overnight, centrifuging, and collecting precipitate to obtain crude pure LOS.
(3) LOS pure
Adding Tris-MgSO into crude LOS 4 Buffer (containing 2mmol/L MgSO) 4 50mmol/L Tris, pH 7.6) 315ml, and suspending to uniformly disperse LOS;
the resuspended LOS solution is divided into 63ml in equal parts, and the volume ratio is 5: 1.3: 2: 1.1: 1.1: 2, shaking the solution for 10 minutes with a large force, centrifuging the solution to separate layers, respectively sucking the upper phase, discarding the upper phase, and respectively collecting the middle layer and the lower phase; respectively adding Tris-MgSO to the intermediate layer solution 4 And (4) adding the buffer solution to 63ml, and then adding the buffer solution into the buffer solution in a volume ratio of 5: 1.3: 2: 1.1: 1.1: 2 in 158ml of a chloroform-methanol solutionExtracting for the second time, and collecting the lower phase.
Respectively combining the two extracted lower phases of different extracting agents, and performing rotary evaporation and drying to obtain LOS.
The comparison graph of the electrophoresis of the purified LOS extracted in example 1 and comparative example 3 is shown in FIG. 6.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent should be subject to the appended claims.

Claims (11)

1. A method for extracting and purifying lipooligosaccharide in thallus is characterized by comprising the following steps:
a. collecting dry bacterial sludge;
b. mixing the dry bacterial sludge and an isobutyric acid ammonia water solution, centrifuging, and collecting supernatant, wherein the isobutyric acid ammonia water solution is a mixture of isobutyric acid and concentrated ammonia water, the mass concentration of the concentrated ammonia water is 22-28%, and the volume ratio of the isobutyric acid to the concentrated ammonia water in the isobutyric acid ammonia water solution is (4-6): 3;
c. mixing the supernatant obtained in the step b with ethanol, standing, centrifuging and collecting precipitates;
d. c, extracting the precipitate obtained in the step c by using a chloroform methanol solution, and collecting an organic phase;
e. d, drying the organic phase obtained in the step d;
the volume ratio of chloroform to methanol in the chloroform-methanol solution is (1-2.5) to 1;
in the step b, 8-12 mL of the isobutyric acid ammonia water solution is added into each gram of dry bacterial sludge.
2. The method for extracting and purifying lipooligosaccharide from bacterial cells according to claim 1, wherein the concentration of ethanol in the mixture of the supernatant and ethanol in step c is (75-85) mL/100mL.
3. The method for extracting and purifying lipooligosaccharide from bacterial cells according to claim 1, wherein step d comprises using MgSO-containing material 4 Resuspending the pellet obtained in step c in the buffer of (1), and extracting the resuspension solution with chloroform-methanol solution.
4. The method for extracting and purifying lipooligosaccharide from a bacterial cell according to claim 3, wherein the lipooligosaccharide comprises MgSO 4 The pH of the buffer solution of (3) is 7.4 to 7.8, mgSO 4 The concentration is 1.5 mmol/L-2.5 mmol/L.
5. The method for extracting and purifying lipooligosaccharide from bacterial cells of claim 4, wherein the concentration of MgSO in the dried bacterial sludge per gram is higher than that of MgSO in the dried bacterial sludge 4 The amount of the buffer solution (2) used is 5 to 10mL.
6. The method for extracting and purifying lipooligosaccharide in bacterial cells according to claim 5, wherein the chloroform methanol solution used in step d and the MgSO-containing solution 4 The volume ratio of the buffer solution (1) to (4) is 1.
7. The method for extracting and purifying lipooligosaccharide in bacterial cells according to claim 1, wherein the organic phase is dried by rotary evaporation in step e.
8. The method for extracting and purifying lipooligosaccharide in bacteria according to claim 7, characterized in that the rotary evaporation drying process comprises the steps of adding water to redissolve, dialyzing and vacuum freeze-drying the dried product.
9. The method for extracting and purifying lipooligosaccharide in bacterial cells according to claim 1, wherein the step a comprises:
a1, centrifuging fermentation liquor to collect thalli;
a2, uniformly mixing purified water and thalli, and centrifugally collecting bacterial sludge;
a3, mixing the bacterial sludge obtained in the step a2 with ethanol, and centrifuging to collect bacterial sludge;
and a4, mixing the bacterial sludge obtained in the step a3 with methanol, and centrifuging to collect dry bacterial sludge.
10. The method for extracting and purifying lipooligosaccharide from a bacterial cell according to any one of claims 1 to 9, wherein the bacterial cell is selected from any one or more of bordetella pertussis, escherichia coli, bordetella parapertussis, salmonella, shigella, pseudomonas, neisseria, and haemophilus.
11. A method for extracting and purifying oligosaccharides from bacterial cells, which comprises obtaining lipooligosaccharides by the method for extracting and purifying lipooligosaccharides from bacterial cells according to any one of claims 1 to 10, and hydrolyzing and purifying the lipooligosaccharides.
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Title
Micromethods for Isolation and Structural Characterization of Lipid A, and Polysaccharide Regions of Bacterial Lipopolysaccharides;Alexey Novikov等;《Microbial Toxins》;20170507;第167-186页 *
Nico Marr等.Variability in the Lipooligosaccharide Structure and Endotoxicity among Bordetella pertussis Strains.《The Journal of Infectious Diseases》.2010,第202卷(第12期),第1897-1906页. *
Variability in the Lipooligosaccharide Structure and Endotoxicity among Bordetella pertussis Strains;Nico Marr等;《The Journal of Infectious Diseases》;20101104;第202卷(第12期);第1897-1906页 *
百日咳鲍特菌脂寡糖的制备及寡糖结合物在小鼠体内免疫原性研究;李鑫等;《微生物学免疫学进展》;20180911;第46卷(第5期);第21-27页 *

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