CN117180412B - Acinetobacter baumannii capsular polysaccharide vaccine and preparation method thereof - Google Patents
Acinetobacter baumannii capsular polysaccharide vaccine and preparation method thereof Download PDFInfo
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention provides a baumannii capsular polysaccharide vaccine and a preparation method thereof, and relates to the technical field of baumannii, wherein the preparation method of the baumannii capsular polysaccharide vaccine adopts the technology of sodium deoxycholate for bacterial lysis, low-temperature centrifugation, tangential flow ultrafiltration, enzyme treatment and the like, and combines the purification method of the baumannii capsular polysaccharide, the preparation method has the advantages of simple process, easy control of operation steps, easy realization, suitability for large-scale production, no pollution of reagents adopted in the preparation method, high yield and high purity of the baumannii capsular polysaccharide, and the prepared baumannii capsular polysaccharide vaccine has the characteristics of high immune protection rate and quick onset of action.
Description
Technical Field
The invention relates to the technical field of acinetobacter baumanii, in particular to an acinetobacter baumanii capsular polysaccharide vaccine and a preparation method thereof.
Background
Acinetobacter baumannii (Acinetobacter baumannii) is a gram-negative pathogenic bacterium, which, as a nosocomial pathogen, can cause a variety of infections including pneumonia, sepsis, meningitis, post-traumatic infection, and urinary tract infection. With the widespread use of antibiotics, multiple resistant acinetobacter baumannii has emerged. Worldwide health organization in 2017 issued a global priority list of antibiotic resistant bacteria and listed acinetobacter baumannii as a serious dangerous pathogen. Vaccines are an effective means of controlling acinetobacter baumanii infection, and many acinetobacter baumanii vaccines are still in preclinical research. Several multicomponent candidate antigens such as inactivated whole cells, outer membrane complexes (outer membrane complexes, OMCs), outer membrane vesicles (outer membrane Vesicles, OMVs) and attenuated live cells have been widely studied. However, the complexity of the components of the multicomponent candidate antigen vaccine and the associated safety problems caused by residual Lipopolysaccharide (LPS) remain to be solved. While many subunit vaccines have overcome safety issues, further exploration is still needed, such as: protein antigens are normally blocked by surface polysaccharides, thereby preventing the recognition of them by antibodies.
Capsular polysaccharides (capsular polysaccharide, CPS) are one of the major virulence factors of capsular bacteria, playing an important role in the bacteria's avoidance of the non-specific defenses of the human body. Meanwhile, the capsular polysaccharide is also a protective antigen of specific immune reaction, is a surface antigen with the least structural change of bacteria, has better immunogenicity, and is one of target antigens which are most suitable for being used as vaccine components. Capsular polysaccharides (capsular polysaccharide, CPS) are composed of closely packed repeating oligosaccharide subunits that form a discrete layer on the cell surface that provides protection for bacteria from survival under different environmental conditions, aids the bacteria in evading host immune defenses, and increases resistance to many antibacterial compounds. Acinetobacter baumannii can produce high molecular weight capsular polysaccharides that are encapsulated around the outer membrane. Improper antibiotic treatment can increase the synthesis of capsular polysaccharide, further enhance the virulence of acinetobacter baumannii and increase the difficulty of clinical treatment.
However, the difficulty in separating and purifying capsular polysaccharide from Acinetobacter baumannii is that the capsule expression amount is low, which is far less than that of haemophilus influenzae, neisseria meningitidis, salmonella typhi, streptococcus pneumoniae and the like. Specific purification methods include extraction with ethanol, phenol organic solvents or precipitation with cetyltrimethylammonium bromide (Cetrimonium (bromide), CTAB) which remove part of the bacterial nucleic acids and proteins, and ion exchange chromatography, gel filtration and affinity chromatography are required for further purification of capsular polysaccharides. However, the use of a large amount of organic solvents such as ethanol and phenol causes environmental pollution, the chromatographic packing is expensive, the operation process is time-consuming and labor-consuming, and the purification method using fractional precipitation, ion exchange chromatography, gel filtration and affinity chromatography has very low yield of capsular polysaccharide and cannot meet clinical requirements. Therefore, the biggest obstacle in developing a acinetobacter baumannii capsular polysaccharide vaccine is how to improve the purification technology of capsular polysaccharide so as to improve the purification effect of acinetobacter baumannii capsular polysaccharide, shorten the operation time and reduce the cost, thereby meeting the clinical requirement of preventing acinetobacter baumannii infection.
Disclosure of Invention
The invention aims to provide an acinetobacter baumannii capsular polysaccharide vaccine and a preparation method thereof. The preferred technical solutions of the technical solutions provided by the present invention can produce a plurality of technical effects described below.
In order to achieve the above purpose, the present invention provides the following technical solutions:
in a first aspect, the invention provides a preparation method of a acinetobacter baumannii capsular polysaccharide vaccine, which comprises the following steps:
(1) Culturing Acinetobacter baumannii;
(2) Collecting Acinetobacter baumannii thalli;
(3) Diluting the bacterial liquid by adopting a PBS buffer with the pH of 0.01M and 7.2-7.4, measuring an OD600 value by an ultraviolet-visible spectrophotometry, adjusting the concentration of the Acinetobacter baumannii bacterial liquid to be 3.5-4.5, taking 500 mL bacterial liquid, adding 10% w/v sodium deoxycholate into the Acinetobacter baumannii bacterial liquid until the final concentration of the bacterial liquid is 0.12% w/v, and then placing the bacterial liquid in a shaking table at 25 ℃ for cracking at 120 rpm for 18 h;
(4) Centrifuging after the completion of the pyrolysis, and taking supernatant after centrifuging and passing through a 0.45 μm filter membrane;
(5) Carrying out tangential flow ultrafiltration on the sample solution passing through the filter membrane by using a 100 kDa membrane, concentrating the liquid, then filtering and washing the liquid by using ultrapure water for a plurality of times, concentrating, transferring the concentrated liquid into a freeze-drying cup, and freeze-drying to obtain crude sugar, and preserving the crude sugar for later use;
(6) Preparing an enzyme reaction buffer solution, preparing the obtained crude sugar into a crude polysaccharide reaction buffer solution of 5 mg/mL by using the enzyme reaction buffer solution, adding 3% v/v of double antibody into the crude polysaccharide reaction buffer solution, and refrigerating in a refrigerator to promote dissolution;
(7) Adding omnipotent nuclease into the crude polysaccharide reaction buffer solution in the step (6), and placing the mixture into a shaking table to react at 37 ℃ and 120 rpm;
(8) Preparing 5% w/vSDS and 10 mg/mL proteinase K for later use by using an enzyme reaction buffer solution, adding the prepared SDS into a crude polysaccharide reaction buffer solution until the final concentration of SDS in the mixed solution is 0.5% w/v, adding the prepared proteinase K until the final concentration is 0.5 mg/mL, and reacting at 37 ℃ in a shaking table at 120 rpm;
(9) After the reaction is finished, diluting the reaction system for 10 times, standing at room temperature, centrifuging, and filtering the centrifuged supernatant with a 0.8 mu m filter membrane;
(10) Adding ultra-pure water into the filtered filtrate, diluting the filtrate, performing ultrafiltration concentration by using 100 kDa membrane tangential flow, then performing filtration and washing for a plurality of times by using ultra-pure water, concentrating, transferring into a freeze-drying cup, and freeze-drying to obtain Acinetobacter baumannii capsular polysaccharide for later use; wherein the number average molecular weight of the prepared capsular polysaccharide is 2724 kDa, and the weight average molecular weight is 7411 kDa;
(11) And (3) dissolving refined sugar by using a sterile PBS solution to prepare the acinetobacter baumannii capsular polysaccharide vaccine.
According to a preferred embodiment, in step (1), further comprising:
drawing Acinetobacter baumannii strains on a trypticase soy agar medium plate, and culturing overnight in a constant temperature incubator at 37 ℃;
selecting single colony to inoculate in trypticase liquid culture medium, culturing in shaking table at 37 deg.c and 220 rpm;
the bacterial liquid obtained by the culture is prepared according to the following ratio of 1:100 proportion was inoculated in trypticase soy broth and incubated overnight at 37℃and 220rpm in a shaker.
According to a preferred embodiment, in step (2), further comprising: the bacterial liquid cultured overnight is centrifugated at 4 ℃ and 4000g for 30min, the supernatant is discarded, and Acinetobacter baumannii bacterial cells are collected.
According to a preferred embodiment, in step (6), further comprising: the enzyme reaction buffer solution comprises Tris-HCl and MgCl with pH of 8.0 2 And CaCl 2 。
In a second aspect, the invention also provides a acinetobacter baumannii capsular polysaccharide vaccine, which is prepared by the preparation method.
In a third aspect, the invention also provides a preparation method of acinetobacter baumannii capsular polysaccharide, which comprises the following steps:
(1) Culturing Acinetobacter baumannii;
(2) Collecting Acinetobacter baumannii thalli;
(3) Diluting the bacterial liquid by adopting a PBS buffer with the pH of 0.01M and 7.2-7.4, measuring an OD600 value by an ultraviolet-visible spectrophotometry, adjusting the concentration of the Acinetobacter baumannii bacterial liquid to be 3.5-4.5, taking 500 mL bacterial liquid, adding 10% w/v sodium deoxycholate into the Acinetobacter baumannii bacterial liquid until the final concentration of the bacterial liquid is 0.12% w/v, and then placing the bacterial liquid in a shaking table at 25 ℃ for cracking at 120 rpm for 18 h;
(4) Centrifuging after the completion of the pyrolysis, and taking supernatant after centrifuging and passing through a 0.45 μm filter membrane;
(5) Carrying out tangential flow ultrafiltration on the sample solution passing through the filter membrane by using a 100 kDa membrane, concentrating the liquid, then filtering and washing the liquid by using ultrapure water for a plurality of times, concentrating, transferring the concentrated liquid into a freeze-drying cup, and freeze-drying to obtain crude sugar, and preserving the crude sugar for later use;
(6) Preparing an enzyme reaction buffer solution, preparing the obtained crude sugar into a crude polysaccharide reaction buffer solution of 5 mg/mL by using the enzyme reaction buffer solution, adding 3% v/v of double antibody into the crude polysaccharide reaction buffer solution, and refrigerating in a refrigerator to promote dissolution;
(7) Adding omnipotent nuclease into the crude polysaccharide reaction buffer solution in the step (6), and placing the mixture into a shaking table to react at 37 ℃ and 120 rpm;
(8) Preparing 5% w/vSDS and 10 mg/mL proteinase K for later use by using an enzyme reaction buffer solution, adding the prepared SDS into a crude polysaccharide reaction buffer solution until the final concentration of SDS in the mixed solution is 0.5% w/v, adding the prepared proteinase K until the final concentration is 0.5 mg/mL, and reacting at 37 ℃ in a shaking table at 120 rpm;
(9) After the reaction is finished, diluting the reaction system for 10 times, standing at room temperature, centrifuging, and filtering the centrifuged supernatant with a 0.8 mu m filter membrane;
(10) Adding ultra-pure water into the filtered filtrate, diluting the filtrate, performing ultrafiltration concentration by using 100 kDa membrane tangential flow, then performing filtration and washing for a plurality of times by using ultra-pure water, concentrating, transferring into a freeze-drying cup, and freeze-drying to obtain Acinetobacter baumannii capsular polysaccharide for later use; wherein the number average molecular weight of the prepared capsular polysaccharide is 2724 kDa, and the weight average molecular weight is 7411 kDa.
Based on the technical scheme, the acinetobacter baumannii capsular polysaccharide vaccine and the preparation method thereof have at least the following beneficial technical effects:
the preparation method of the Acinetobacter baumannii capsular polysaccharide vaccine is simple in process, easy to control and realize in operation steps, suitable for large-scale production, free of pollution by reagents adopted in the preparation method, and 1.67 times of the yield of capsular polysaccharide purified by an ethanol precipitation method is obtained by an enzyme treatment method. The Acinetobacter baumannii capsular polysaccharide prepared by the enzyme treatment method has high yield and high purity. The prepared acinetobacter baumannii capsular polysaccharide vaccine has the characteristics of high immune protection rate and quick response.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a molecular weight diagram of an African blue staining method for detecting the capsular polysaccharide of Acinetobacter baumannii in the embodiment of the invention, wherein M represents Marker, and 1 and 2 are respectively capsular polysaccharide repeated groups;
FIG. 2 is a graph of laser light scattering signals of Acinetobacter baumannii capsular polysaccharide according to an embodiment of the present invention;
FIG. 3 is a graph showing the particle size distribution of Acinetobacter baumannii capsular polysaccharide according to an embodiment of the present invention;
FIG. 4 is a FT-IR spectrum of Acinetobacter baumannii capsular polysaccharide according to an embodiment of the invention;
FIG. 5 is a 400 MHz view of Acinetobacter baumannii capsular polysaccharide according to an embodiment of the invention 1 H NMR detection spectrum, wherein the solvent is heavy water;
FIG. 6 is a graph of survival rate of mice vaccinated with the Acinetobacter baumannii polysaccharide vaccine of the present invention after challenge;
FIG. 7 is a graph of survival rate of mice vaccinated with the Acinetobacter baumannii polysaccharide vaccine of the present invention once after challenge.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, based on the examples herein, which are within the scope of the invention as defined by the claims, will be within the scope of the invention as defined by the claims.
Example 1
The embodiment 1 of the application provides a preparation method of acinetobacter baumannii capsular polysaccharide.
1. Extraction of acinetobacter baumannii capsular polysaccharide.
Bacterial strain and reagent sources: acinetobacter baumannii ATCC17978 was purchased from the American type culture Collection (American Type Culture Collection, ATCC). Proteinase K and alisxin blue staining (pH 2.5) were purchased from Soy Corp, sodium deoxycholate, PBS was purchased from Milin reagent, BCA protein quantification kit was purchased from Biyun biological, tryptic Soy Agar solid media (TSA) and Tryptic Soy broth media (Tryptic Soy broth, TSB) were purchased from Beijing aoboxing Biotechnology Limited. Totipotent nucleases were purchased from the following holy biology company. 100 The kDa membrane package was purchased from Sidoris corporation.
The method comprises the following steps:
(1) Culturing Acinetobacter baumannii ATCC17978:
acinetobacter baumannii ATCC17978 was streaked onto TSA plates and incubated overnight in a constant temperature incubator at 37 ℃.
Single colonies were selected and inoculated into 10mL of TSB broth and incubated in a shaker at 37℃and 220rpm for 5 hours.
The bacterial liquid obtained by the culture is prepared according to the following ratio of 1: 100% was inoculated into 1L of TSB liquid medium and incubated overnight at 37℃and 220rpm in a shaker.
(2) Centrifuging at 4deg.C for 30min at 4000 and g, removing supernatant, and collecting Acinetobacter baumannii thallus.
(3) Diluting the bacterial liquid with 0.01M PBS (pH 7.2-7.4), measuring the OD600 value by ultraviolet-visible spectrophotometry, regulating the bacterial liquid concentration to 3.5-4.5, taking 500 mL bacterial liquid, adding 10% w/v sodium Deoxycholate (DOC) to the bacterial liquid of Acinetobacter baumannii to a final concentration of 0.12% w/v, and then placing in a shaking table at 25 ℃ for 18 h under 120 rpm. The cracking conditions can ensure that the crude polysaccharide has good solubility in the enzymolysis buffer solution in the step (7).
(4) After completion of the lysis, the mixture was centrifuged at 14000g at 4℃for 5min and 1 time, and the supernatant was filtered through a 0.45 μm filter to remove insoluble particles.
(5) Taking a sample solution after 500 mL filtration membrane, carrying out tangential flow ultrafiltration on the sample solution by using a 100 kDa membrane, concentrating the solution to 100 mL, filtering and washing the solution by using ultrapure water for three times, filtering and washing the solution by using 100 mL ultrapure water each time, concentrating the solution to 20 mL, pouring out the residual liquid in the pipeline, rinsing the solution by using ultrapure water, collecting the rinsed liquid, transferring the rinsed liquid into a freeze-drying cup, and freeze-drying the solution to obtain 160 mg crude sugar, and storing the 160 crude sugar in a refrigerator at the temperature of minus 80 ℃. The nucleic acid content and the protein content in the crude sugar were measured by the Nanodrop and BCA (Bicinchoninic Acid) protein concentration measurement methods and were 10.52% and 26.52%, respectively.
(6) Preparing enzyme reaction buffer solution, wherein the components of the prepared enzyme reaction buffer solution are 100 mM Tris-HCl and 2 mM MgCl with pH of 8.0 2 And 5 mM CaCl 2 。
(7) A crude polysaccharide solution at a concentration of 5 mg/mL was prepared with enzyme reaction buffer, and after adding 3% v/v of the diabody (penicillin and streptomycin, gibco), the solution was placed in a refrigerator at 4℃for 3 h to promote solubilization. As the molecular weight of the capsular polysaccharide is large, a 0.22 mu m filter membrane cannot be adopted for sterilization, and the addition of the double antibody can effectively avoid bacterial pollution in the nuclease treatment and protease treatment processes.
(8) To 20 mL of the crude polysaccharide reaction buffer, 0.8. Mu.L of a omnipotent nuclease (UCF.ME.UltraNuclease omnipotent nuclease (same as Benzonase)) was added, and reacted at 37℃in a shaker at 120 rpm for 15 h.
(9) 5% w/v SDS and 10 mg/mL proteinase K (Soy) were prepared for use in the enzyme reaction buffer, SDS was added to the crude polysaccharide reaction buffer to a final SDS concentration of 0.5% w/v, proteinase K was added to a final SDS concentration of 0.5 mg/mL, and reacted at 37℃in a shaker at 120 rpm for 24 h.
(10) After the reaction, the reaction system was diluted 10 times, left at room temperature for 30min, centrifuged at 4000g for 5min at 4℃and once, and the supernatant was filtered through a 0.8 μm filter.
(11) Adding 200-mL (room temperature) ultrapure water into the filtered filtrate 200-mL, diluting the filtrate, performing ultrafiltration concentration to 80-mL by using a 100 kDa membrane tangential flow, filtering and washing with the ultrapure water three times, filtering and washing with 160-mL ultrapure water each time, concentrating to 10-20-mL, pouring out the liquid remained in the pipeline, rinsing with the ultrapure water, collecting the rinsed liquid, transferring into a freeze-drying cup, performing freeze-drying to obtain 15-mg refined sugar, and storing in a refrigerator at-80 ℃. The content of nucleic acid and protein in refined sugar was 2.4% and 4.2%, respectively. Compared with crude polysaccharide, the removal rate of nucleic acid and protein after treatment by omnipotent nuclease and proteinase K reaches 77.19% and 84.16%, respectively, and the loss of capsular polysaccharide is less, so that refined sugar with higher purity and yield can be obtained.
2. The sugar content of the capsular polysaccharide was measured by the sulfuric acid-phenol method.
Weighing a small amount of refined sugar, preparing a solution with the concentration of 1 mg/mL by using ultrapure water, and diluting the solution to 0.2 mg/mL by using the purified water for later use. A small amount of glucose was weighed and prepared into a 1. 1 mg/mL solution with ultrapure water, and then diluted to a final concentration of 0.2 mg/mL solution for later use. Glucose solution 0, 75. Mu.L, 150. Mu.L, 225. Mu.L, 300. Mu.L, 375. Mu.L and 450. Mu.L are respectively taken out from an EP tube, supplemented to 0.5. 0.5 mL by ultrapure water, concentrated sulfuric acid 5 mL is added, the mixture is uniformly mixed and then reacted in a water bath at 80 ℃ for 60 min, 200. Mu.L is taken out, 5% phenol 40. Mu.L is added, the reaction is carried out at room temperature for 20 min, the absorbance value is measured at 490 nm, and a standard curve is drawn. Taking 450 mu L of refined water solution, supplementing the refined water solution to 0.5 mL by ultrapure water, adding concentrated sulfuric acid 5 mL, uniformly mixing, respectively reacting in a water bath at 80 ℃ for 140 min, taking out 200 mu L, adding 5% phenol 40 mu L, reacting at room temperature for 20 min, and measuring the absorbance value at 490 nm. Substituting the absorbance value of refined sugar into a standard curve prepared from glucose solution, the sugar content in refined sugar is 48.73%.
3. The molecular weight of the acinetobacter baumannii capsular polysaccharide is detected by an aliskiren blue staining method.
The capsular polysaccharide was mixed with SDS-PAGE protein loading buffer (5X) and boiled for 10 min, the loading of capsular polysaccharide in each lane was 30. Mu.g, and after SDS-PAGE gel electrophoresis was completed, the gel was incubated in a destaining solution (destaining solution component 25% v/v ethanol, 10% v/v acetic acid solution) at 50℃for 5, 10, 15min, respectively. In dark conditions, the gel was stained with an aliskiren blue staining solution (pH 2.5), soxhobao) at 50℃for 15 min. And finally adding the decolorized solution into a horizontal shaking table or a side shaking table for slow shaking and eluting. After destaining, the gel was photographed using a protein gel imaging method. As shown in FIG. 1, the capsular polysaccharide obtained by the above method has a molecular weight of 72 kDa or more. Because alisxin blue can form insoluble complexes with acidic glycosaminoglycans, the resulting capsular polysaccharide contains acidic glycosaminoglycans.
4. And (5) detecting the molecular weight of the acinetobacter baumannii capsular polysaccharide.
To determine the molecular weight of the isolated acinetobacter baumanii capsular polysaccharide of the present application, the molecular weight of the acinetobacter baumanii capsular polysaccharide was detected by gel permeation chromatography (Gel Permeation Chromatography, GPC). As shown in FIG. 2, the number average molecular weight (M n ) 2724 kDa, weight average molecular weight (M w ) The polydispersity PDI is 2.72 and the molecular weight distribution is broad at 7411 kDa.
5. Particle size of acinetobacter baumannii.
The particle size of the Acinetobacter baumannii capsular polysaccharide was measured by a Markov particle size potentiometer (Zetasizer Pro Blue), and the Acinetobacter baumannii capsular polysaccharide was dissolved in pure water to prepare a solution having a concentration of 1 mg/mL. As shown in FIG. 3, the average particle size of Acinetobacter baumannii capsular polysaccharide was 735.9 nm, PDI was 0.33, and average surface potential was-27.13. 27.13 mV. Since the particle size distribution of the acinetobacter baumannii capsular polysaccharide is wide and the average particle size is larger than 220 and nm, most capsular polysaccharide cannot pass through a 0.22 μm filter membrane, so that capsular polysaccharide cannot pass through a 0.22 μm filter membrane for filter sterilization.
6. Structural analysis of acinetobacter baumannii capsular polysaccharide.
To determine the structure of capsular polysaccharides, the near infrared spectrum of capsular polysaccharides was studied, as shown in FIG. 4, at 3298 cm −1 There is a strong and broad absorption peak, which is the stretching vibration peak of OH. At 2926 cm -1 A characteristic peak of carbohydrate is observed, suggesting the presence of stretching vibration of CH bonds in the capsular polysaccharide. 1660 cm -1 And 1558 cm -1 The strong absorption peak at this point indicates the presence of N-linked acetyl groups in the Acinetobacter baumannii capsular polysaccharide. At 1200-1000 cm -1 Absorption peak in the range (1132 and cm) -1 ，1060 cm -1 ) The presence of a pyran ring was demonstrated. 832 cm −1 And 892 cm −1 Weak suction at the siteThe peak recovery indicates the presence of monosaccharides in both the alpha and beta configuration in the polysaccharide.
Capsular polysaccharide is subjected to 1 H-NMR testing to analyze the monosaccharide classes that may be present in the capsular polysaccharide. As shown in FIG. 5, δ5.01 may be the chemical shift of the proton of the α -N-acetamido (NAc) end group of fucose (FucNAc), δ4.87 ppm may be the chemical shift of the proton of the β -N-acetamido (NAc) glucose (GlcNAc) end group, chemical shift 1.96-2.04 ppm is the NAc signal, δ3.3-4.5 is the cyclic sub-region, and δ1.2- δ1.3 ppm may be the methyl signal on fucose.
7. Immune protection effect of acinetobacter baumannii capsular polysaccharide vaccine.
For studying the protective effect of a. Baumannii capsular polysaccharide vaccine, female C57bl/6 mice, 7-9 weeks old, and 17-19 g in weight, were selected and randomly divided into PBS group (control group) and CPS group (vaccine group), each group of 5 mice. The capsular polysaccharide was dissolved in 0.01M sterile PBS (pH 7.2-7.4), a vaccine preparation at a concentration of 1.5. 1.5 mg/mL was prepared, and C57BL/6 mice were subjected to nasal drip immunization on days 0 and 21, the volume of the nasal drip vaccine preparation was 20. Mu.L per mouse, and the dose of the capsular polysaccharide vaccine was 30. Mu.g per mouse. On day 28, live acinetobacter baumannii bacteria were used for tracheal intubation to attack toxins, and survival rates of mice were observed within one week. As shown in table 1 and fig. 6, mice immunized with the capsular polysaccharide group had 100% survival, while mice immunized with PBS group had 0% survival. The result shows that the capsular polysaccharide has high immune protection rate, and can effectively prevent the infection of Acinetobacter baumannii after the animal is immunized in vivo.
TABLE 1 survival of mice within one week after challenge with live Acinetobacter baumannii
In another set of parallel experiments, we performed nasal drop immunization of C57BL/6 mice on day 0, each at a dose of 30 μg capsular polysaccharide. After 7 days of immunization, live acinetobacter baumannii is used for tracheal intubation to attack toxin, and the survival rate of the mice is observed within one week. As shown in table 2 and fig. 7, mice immunized with the capsular polysaccharide group had 100% survival, while mice immunized with PBS group had 0% survival. The immunization program of the prior art is immunization on day 0, day 14 and day 28, and live bacterial challenge on day 70. Compared with the existing immunization program, the experiment of the application carries out live bacteria detoxification on the seventh day after immunization, and the protection rate of mice reaches 100%, which shows that the immune protection capability of the purified Acinetobacter baumannii capsular polysaccharide has the characteristic of quick effect, and the prepared capsular polysaccharide has larger molecular weight and can be rapidly identified by immune cells.
TABLE 2 survival of mice after one vaccination of mice with one week after challenge with live Acinetobacter baumannii
The acinetobacter baumannii capsular polysaccharide adopts a purification method combining technologies such as deoxycholate sodium bacteria lysis, low-temperature centrifugation, tangential flow ultrafiltration, enzyme treatment and the like, and the purification method does not need an organic solvent and expensive chromatographic medium, so that the environmental pollution is reduced, the production period is shortened, the cost is low, the preparation method is suitable for large-scale production, and the prepared acinetobacter baumannii capsular polysaccharide vaccine has an efficient immune protection effect.
Example 2
The embodiment 2 also provides a preparation method of the acinetobacter baumannii capsular polysaccharide by adopting the ethanol precipitation method, which comprises the following steps:
2.1 Acinetobacter baumannii ATCC17978 was taken, streaked onto TSA plates and incubated overnight in a 37℃incubator.
2.2, select single colony inoculated in 10mL TSB liquid medium, in a shaking table at 37 ℃,220rpm culture for 5 hours.
2.3, the bacterial liquid obtained by the culture is prepared according to the following formula 1: 100% was inoculated in 1L of TSB broth and incubated overnight at 37℃and 220rpm in a shaker.
2.4, centrifuging at 4 ℃ for 30min at 4000g, discarding the supernatant, and collecting LAC-4 thalli.
2.5, diluting the bacterial solution with 0.01M PBS (pH 7.2-7.4), measuring the OD600 value, adjusting the bacterial solution concentration to 3.5-4.5, taking 500 mL bacterial solution, adding 10% w/v sodium Deoxycholate (DOC) to the final concentration of 0.12% into LAC-4 bacterial cells, and then placing in a shaking table at 25 ℃ for 18 h cleavage at 120 rpm.
2.6, after complete lysis, centrifuging at 14000g for 5min at 4deg.C for 1 time, and collecting supernatant, and filtering with 0.45 μm filter membrane.
2.7, taking a sample solution after a 500 mL filtration membrane, carrying out tangential flow ultrafiltration on the sample solution by using a 100 kDa membrane, concentrating the solution to 100 mL, filtering and washing the solution by using ultrapure water three times, filtering and washing the solution by using 100 mL ultrapure water each time, concentrating the solution to 20 mL, pouring out the liquid remained in the pipeline, rinsing the solution by using ultrapure water, collecting the rinsed liquid, and combining the collected liquid to obtain the liquid of 50 mL.
2.8, taking an ultrafiltration concentrated sample, and adding CaCl 2 The solution was brought to a final concentration of 0.5mol/L, and after mixing well, absolute ethanol was added to a final concentration of 25%, and the mixture was left to stand overnight in a refrigerator at 4 ℃.
2.9, centrifuging the sample obtained in the step 2.8 at 4 ℃ for 15min with 14000g, discarding the precipitate, taking the supernatant, adding absolute ethanol to a final concentration of 80%, and placing in a refrigerator to precipitate.
2.10, centrifuging the sample from the step 2.9 at 4 ℃ and 14000g for 15min, discarding the supernatant, dissolving the precipitate with a small amount of ultrapure water, storing in a refrigerator at-80 ℃ and freeze-drying to obtain the 9 mg capsular polysaccharide. The content of capsular polysaccharide obtained by ethanol precipitation is relatively small compared to the preparation method in example 1 of the present application.
The nucleic acid content and the protein content of the capsular polysaccharide obtained by the ethanol precipitation method were 16.6% and 17.1% respectively by using a BCA (Bicinchoninic Acid) protein concentration determination method and a Nanodrop determination method.
The capsular polysaccharide obtained by a small amount of ethanol precipitation method is weighed, prepared into a solution with the concentration of 1 mg/mL by using ultrapure water, and then diluted to 0.2 mg/mL for standby. A small amount of glucose was weighed and prepared into a 1. 1 mg/mL solution with ultrapure water, and then diluted to a final concentration of 0.2 mg/mL solution for later use. Glucose solutions 0, 75, 150, 225, 300, 375 and 450. Mu.L were taken in EP tubes, made up to 0.5. 0.5 mL with ultrapure water, added with concentrated sulfuric acid 5 mL, mixed well, reacted in a 80℃water bath for 60 min, taken out 200. Mu.L, added with 5% phenol 40. Mu.L, reacted at room temperature for 20 min, and absorbance was measured at 490 nm, and a standard curve was drawn. Taking 450 mu L of refined water solution, supplementing the refined water solution to 0.5 mL by ultrapure water, adding concentrated sulfuric acid 5 mL, uniformly mixing, respectively reacting in an 80 ℃ water bath for 140 min, taking out 200 mu L, adding 5% phenol 40 mu L, reacting for 20 min at room temperature, and measuring the absorbance value at 490 nm. Substituting the absorbance value of refined sugar into a standard curve prepared by glucose solution to obtain the capsular polysaccharide with 20.52% sugar content by ethanol precipitation method.
Compared with the ethanol precipitation method, the preparation method of the embodiment 1 of the application has the advantages that the yield of the capsular polysaccharide obtained by the enzyme treatment method is 1.67 times that of the capsular polysaccharide purified by the ethanol precipitation method, the nucleic acid content and the protein content of the capsular polysaccharide obtained by the enzyme treatment method are lower than those of the capsular polysaccharide obtained by the ethanol precipitation method, the measured sugar content is higher, the purity is higher, and the preparation method of the application can remarkably improve the yield and the purity of the acinetobacter baumannii capsular polysaccharide.
The foregoing is merely illustrative of the present invention, and the present invention is not limited thereto, and any person skilled in the art will readily recognize that variations or substitutions are within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (6)
1. The preparation method of the acinetobacter baumannii capsular polysaccharide vaccine is characterized by comprising the following steps of:
(1) Culturing Acinetobacter baumannii ATCC17978;
(2) Collecting Acinetobacter baumannii thalli;
(3) Diluting the bacterial liquid by adopting a PBS buffer with the pH of 0.01M and 7.2-7.4, measuring an OD600 value by an ultraviolet-visible spectrophotometry, adjusting the concentration of the Acinetobacter baumannii bacterial liquid to be 3.5-4.5, taking 500 mL bacterial liquid, adding 10% w/v sodium deoxycholate into the Acinetobacter baumannii bacterial liquid until the final concentration of the bacterial liquid is 0.12% w/v, and then placing the bacterial liquid in a shaking table at 25 ℃ for cracking at 120 rpm for 18 h;
(4) Centrifuging after the completion of the pyrolysis, and taking supernatant after centrifuging and passing through a 0.45 μm filter membrane;
(5) Carrying out tangential flow ultrafiltration on the sample solution passing through the filter membrane by using a 100 kDa membrane, concentrating the liquid, then filtering and washing the liquid by using ultrapure water for a plurality of times, concentrating, transferring the concentrated liquid into a freeze-drying cup, and freeze-drying to obtain crude sugar, and preserving the crude sugar for later use;
(6) Preparing an enzyme reaction buffer solution, preparing the obtained crude sugar into a crude polysaccharide reaction buffer solution of 5 mg/mL by using the enzyme reaction buffer solution, adding 3% v/v of double antibody into the crude polysaccharide reaction buffer solution, and refrigerating in a refrigerator to promote dissolution;
(7) Adding omnipotent nuclease into the crude polysaccharide reaction buffer solution in the step (6), and placing the mixture into a shaking table to react at 37 ℃ and 120 rpm;
(8) Preparing 5% w/vSDS and 10 mg/mL proteinase K for later use by using an enzyme reaction buffer solution, adding the prepared SDS into a crude polysaccharide reaction buffer solution until the final concentration of SDS in the mixed solution is 0.5% w/v, adding the prepared proteinase K until the final concentration is 0.5 mg/mL, and reacting at 37 ℃ in a shaking table at 120 rpm;
(9) After the reaction is finished, diluting the reaction system for 10 times, standing at room temperature, centrifuging, and filtering the centrifuged supernatant with a 0.8 mu m filter membrane;
(10) Adding ultra-pure water into the filtered filtrate, diluting the filtrate, performing ultrafiltration concentration by using 100 kDa membrane tangential flow, then performing filtration and washing for a plurality of times by using ultra-pure water, concentrating, transferring into a freeze-drying cup, and freeze-drying to obtain Acinetobacter baumannii capsular polysaccharide for later use; wherein the number average molecular weight of the prepared capsular polysaccharide is 2724 kDa, and the weight average molecular weight is 7411 kDa;
(11) And (3) dissolving refined sugar by using a sterile PBS solution to prepare the acinetobacter baumannii capsular polysaccharide vaccine.
2. The method according to claim 1, wherein in step (1), further comprising:
acinetobacter baumannii ATCC17978 strain is taken, streaked on a trypticase soy agar medium plate, and cultured overnight in a constant temperature incubator at 37 ℃;
selecting single colony to inoculate in trypticase liquid culture medium, culturing in shaking table at 37 deg.c and 220 rpm;
the bacterial liquid obtained by the culture is prepared according to the following ratio of 1:100 proportion was inoculated in trypticase soy broth and incubated overnight at 37℃and 220rpm in a shaker.
3. The method according to claim 2, wherein in step (2), further comprising: the bacterial liquid cultured overnight is centrifugated at 4 ℃ and 4000g for 30min, the supernatant is discarded, and Acinetobacter baumannii bacterial cells are collected.
4. The method of claim 1, wherein in step (6), further comprising: the enzyme reaction buffer solution comprises Tris-HCl and MgCl with pH of 8.0 2 And CaCl 2 。
5. An acinetobacter baumannii capsular polysaccharide vaccine, characterized in that said acinetobacter baumannii capsular polysaccharide vaccine is prepared by the preparation method of any of the preceding claims 1 to 4.
6. The preparation method of the acinetobacter baumannii capsular polysaccharide is characterized by comprising the following steps of:
(1) Culturing Acinetobacter baumannii ATCC17978;
(2) Collecting Acinetobacter baumannii thalli;
(3) Diluting the bacterial liquid by adopting a PBS buffer with the pH of 0.01M and 7.2-7.4, measuring an OD600 value by an ultraviolet-visible spectrophotometry, adjusting the concentration of the Acinetobacter baumannii bacterial liquid to be 3.5-4.5, taking 500 mL bacterial liquid, adding 10% w/v sodium deoxycholate into the Acinetobacter baumannii bacterial liquid until the final concentration of the bacterial liquid is 0.12% w/v, and then placing the bacterial liquid in a shaking table at 25 ℃ for cracking at 120 rpm for 18 h;
(4) Centrifuging after the completion of the pyrolysis, and taking supernatant after centrifuging and passing through a 0.45 μm filter membrane;
(5) Carrying out tangential flow ultrafiltration on the sample solution passing through the filter membrane by using a 100 kDa membrane, concentrating the liquid, then filtering and washing the liquid by using ultrapure water for a plurality of times, concentrating, transferring the concentrated liquid into a freeze-drying cup, and freeze-drying to obtain crude sugar, and preserving the crude sugar for later use;
(6) Preparing an enzyme reaction buffer solution, preparing the obtained crude sugar into a crude polysaccharide reaction buffer solution of 5 mg/mL by using the enzyme reaction buffer solution, adding 3% v/v of double antibody into the crude polysaccharide reaction buffer solution, and refrigerating in a refrigerator to promote dissolution;
(7) Adding omnipotent nuclease into the crude polysaccharide reaction buffer solution in the step (6), and placing the mixture into a shaking table to react at 37 ℃ and 120 rpm;
(8) Preparing 5% w/vSDS and 10 mg/mL proteinase K for later use by using an enzyme reaction buffer solution, adding the prepared SDS into a crude polysaccharide reaction buffer solution until the final concentration of SDS in the mixed solution is 0.5% w/v, adding the prepared proteinase K until the final concentration is 0.5 mg/mL, and reacting at 37 ℃ in a shaking table at 120 rpm;
(9) After the reaction is finished, diluting the reaction system for 10 times, standing at room temperature, centrifuging, and filtering the centrifuged supernatant with a 0.8 mu m filter membrane;
(10) Adding ultra-pure water into the filtered filtrate, diluting the filtrate, performing ultrafiltration concentration by using 100 kDa membrane tangential flow, then performing filtration and washing for a plurality of times by using ultra-pure water, concentrating, transferring into a freeze-drying cup, and freeze-drying to obtain Acinetobacter baumannii capsular polysaccharide for later use; wherein the number average molecular weight of the prepared capsular polysaccharide is 2724 kDa, and the weight average molecular weight is 7411 kDa.
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