阅读说明：本技术 b型流感嗜血杆菌荚膜多糖制备方法 (Preparation method of type b haemophilus influenzae capsular polysaccharide ) 是由 胡浩 刘佳 朱学军 沈向向 于旭博 于 2020-12-04 设计创作，主要内容包括：本发明涉及生物技术领域,具体而言,涉及一种b型流感嗜血杆菌荚膜多糖制备方法。该方法包括对发酵澄清液进行超滤浓缩得到第一浓缩液；将所述第一浓缩液与除核酸液共搅拌后,离心收集上清,超滤浓缩得到第二浓缩液；所述除核酸液为EDTA、醋酸钠和脱氧胆酸钠的30％～50％(v/v)的乙醇溶液；向所述第二浓缩液中加入CTAB至终浓度0.5％～1.5％(g/ml)共搅拌以提取荚膜多糖,离心收集沉淀,用65％～75％(v/v)的乙醇复溶沉淀使得所述荚膜多糖溶剂其中得到复合多糖提取液；对所述复合多糖提取液离心并收集上清,使用无机盐解离除去CTAB后醇沉荚膜多糖。(The invention relates to the technical field of biology, and particularly relates to a preparation method of a type b haemophilus influenzae capsular polysaccharide. The method comprises the steps of carrying out ultrafiltration concentration on fermented clarified liquid to obtain a first concentrated solution; after the first concentrated solution and the nucleic acid removing solution are stirred together, centrifuging and collecting supernatant, and performing ultrafiltration concentration to obtain a second concentrated solution; the nucleic acid removing liquid is 30-50% (v/v) ethanol solution of EDTA, sodium acetate and sodium deoxycholate; adding CTAB into the second concentrated solution to a final concentration of 0.5-1.5% (g/ml) and stirring to extract capsular polysaccharide, centrifuging to collect precipitate, and re-dissolving the precipitate with 65-75% (v/v) ethanol to obtain a compound polysaccharide extracting solution; and centrifuging the composite polysaccharide extracting solution, collecting supernatant, dissociating by using inorganic salt to remove CTAB, and then precipitating capsular polysaccharide by alcohol.)

A preparation method of a Haemophilus influenzae type b capsular polysaccharide is characterized by comprising the following steps:

a) carrying out ultrafiltration concentration on the fermented clarified liquid to obtain a first concentrated liquid;

b) adding a nucleic acid removing liquid into the first concentrated solution, stirring, centrifuging, collecting supernatant, and performing ultrafiltration concentration to obtain a second concentrated solution;

the nucleic acid removing liquid is 30-50% (v/v) ethanol solution containing 0.001-0.003 mol/L EDTA, 4-8% (g/ml) sodium acetate and 0.5-1.5% (g/ml) sodium deoxycholate;

the volume of the first concentrated solution and the second concentrated solution is independently selected from 1/10-1/3 of the volume of the fermentation clarified solution;

c) adding CTAB into the second concentrated solution to a final concentration of 0.5-1.5% (g/ml) for incubation so as to extract capsular polysaccharide, stirring, centrifuging, collecting precipitate, and re-dissolving the precipitate with 65-75% (v/v) ethanol so as to dissolve the capsular polysaccharide to obtain a composite polysaccharide extracting solution;

d) and centrifuging the composite polysaccharide extracting solution, collecting supernatant, dissociating by using inorganic salt to remove CTAB, and then precipitating capsular polysaccharide by alcohol.

2. The process for preparing the haemophilus influenzae type b capsular polysaccharide according to claim 1, further comprising step e) after step d):

redissolving the capsular polysaccharide obtained by alcohol precipitation with water, ultrafiltering and concentrating, collecting the trapped fluid and drying.

3. The method for preparing the haemophilus influenzae type b capsular polysaccharide according to claim 1, wherein the volume ratio of the fermentation concentrate to the nucleic acid removing solution is 1: (1.5-2.5).

4. The method for preparing the Haemophilus influenzae type b capsular polysaccharide according to claim 3, wherein dissociating to remove CTAB comprises:

adding sodium chloride into the supernatant to a final concentration of 0.4-0.6M, stirring for 30min at 2-8 ℃, centrifuging, and collecting the precipitate;

dissolving the precipitate in 30-50% (v/v) ethanol containing 0.3-0.5M sodium chloride, and centrifuging to collect the supernatant.

5. The preparation method of Haemophilus influenzae type b capsular polysaccharide according to any one of claims 1-4, wherein the molecular weight cut-off of the membrane used for ultrafiltration concentration is 90 k-110 k.

6. The preparation method of Haemophilus influenzae type b capsular polysaccharide according to any one of claims 1-4, wherein the stirring time in step b) is 2-4 h.

7. The method for preparing the Haemophilus influenzae type b capsular polysaccharide according to any one of claims 1-4, wherein the stirring time in step c) is 4-8 h.

8. The preparation method of the Haemophilus influenzae type b capsular polysaccharide according to any one of claims 1-4, wherein the redissolving and precipitation with 65-75% (v/v) ethanol is performed under stirring for 8-16 h.

9. The preparation method of the Haemophilus influenzae type b capsular polysaccharide according to any one of claims 1-4, wherein the serotype of Haemophilus influenzae type b is selected from one or more of a, b, c, d, e and f.

10. A composition containing a haemophilus influenzae type b capsular polysaccharide prepared by a process as claimed in any one of claims 1 to 9.

11. A vaccine comprising the composition of claim 10.

Technical Field

The invention relates to the technical field of biology, and particularly relates to a preparation method of a type b haemophilus influenzae capsular polysaccharide.

Background

Haemophilus influenzae type b, abbreviated as Hib, is a common commensal bacterium in children's nasopharynx, and can cause severe pneumonia, meningitis and other invasive diseases. Almost all patients are children under 5 years of age, with children aged 4 to 18 months being particularly fragile. It is transmitted from infected individuals to susceptible individuals via the respiratory tract. Haemophilus influenzae type B also causes potentially serious inflammatory infections of the face, mouth, blood, epiglottis, joints, heart, bones, peritoneum, and trachea. Most children carry Hib for some period of time, sometimes months, before the vaccine is applied, but the rate of bacterial colonization varies greatly from socioeconomic status to socioeconomic status depending on age. In areas with high Hib vaccination rate, the colonization rate of bacteria in the nasopharynx is very low due to the group immunization effect of the Hib conjugate vaccine.

Hib can be divided into 6 (a, b, c, d, e, f) serotypes according to the antigenic components of the capsular polysaccharide, and by using type-specific immune sera for capsular swelling tests or other serological tests. Among them, type b is the most pathogenic and most common to infants, type f is the second. The main component of the haemophilus influenzae type b capsular polysaccharide is polyribosyl ribitol phosphate.

Over the last 60 years, various aspects of Hib research have been widely carried out in many countries, and four different types of Hib polysaccharide-protein conjugate vaccines with good immunogenicity have been developed in sequence in the prior art. The sustained high coverage of vaccines has caused a substantial decrease in the incidence of Hib disease in the united states. The generation of the immunity of the crowd is the result of directly protecting children by inoculating the Hib vaccine, and the immunity of the crowd prevents nasopharyngeal bacteria and blocks the transmission of Hib. The use of these vaccines significantly reduces the incidence of Hib meningitis and epiglottitis. Purification of polysaccharides is a very important step in vaccine production processes. According to a method for purifying the b-type haemophilus influenzae capsular polysaccharide provided by the current edition of pharmacopoeia of the people's republic of China, phenol which is a toxic and easily-corroded reagent is adopted, the consumption of CTAB is large, and the preparation cost is high; and the endotoxin is removed by adopting an ultracentrifugation mode, so that the cost is high and the mass production is difficult. In addition, the problems of high protein and nucleic acid content, low ribose purity and the like still exist after purification.

Disclosure of Invention

In order to achieve the above purpose of the present invention, the following technical solutions are adopted:

the invention relates to a preparation method of a type b haemophilus influenzae capsular polysaccharide, which comprises the following steps:

a) carrying out ultrafiltration concentration on the fermented clarified liquid to obtain a first concentrated liquid;

b) adding a nucleic acid removing liquid into the first concentrated solution, stirring, centrifuging, collecting supernatant, and performing ultrafiltration concentration to obtain a second concentrated solution;

the nucleic acid removing liquid is 30-50% (v/v) ethanol solution containing 0.001-0.003 mol/L EDTA, 4-8% (g/ml) sodium acetate and 0.5-1.5% (g/ml) sodium deoxycholate;

the volume of the first concentrated solution and the second concentrated solution is independently selected from 1/10-1/3 of the volume of the fermentation clarified solution;

c) adding CTAB into the second concentrated solution to a final concentration of 0.5-1.5% (g/ml) for incubation so as to extract capsular polysaccharide, stirring, centrifuging, collecting precipitate, and re-dissolving the precipitate with 65-75% (v/v) ethanol so as to dissolve the capsular polysaccharide to obtain a composite polysaccharide extracting solution;

d) and centrifuging the composite polysaccharide extracting solution, collecting supernatant, dissociating by using inorganic salt to remove CTAB, and then precipitating capsular polysaccharide by alcohol.

According to a further aspect of the invention, the invention also relates to the Haemophilus influenzae type b capsular polysaccharide prepared by the method.

The invention also relates to a vaccine containing the composition as described above.

Compared with the prior art, the invention has the beneficial effects that:

according to the invention, the fermentation clarified liquid is subjected to ultrafiltration concentration, so that a part of impurities are removed firstly, the pressure of subsequent impurity removal operation is reduced, the operation volume is reduced, and the operation convenience is improved; the method is characterized in that most of nucleic acid, protein and endotoxin are removed by adding low-concentration ethanol containing EDTA, sodium acetate and DOC with specific concentration into the fermentation concentrated solution, so that the phenol extraction step is replaced, and the operation difficulty and the operation safety risk are reduced; the method has the advantages of high yield, stable output, low impurity content and the like.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a NMR chart of capsular polysaccharide prepared in one example of the invention;

FIG. 2 is a NMR chart of capsular polysaccharide prepared in one example of the invention;

FIG. 3 is a NMR chart of capsular polysaccharide prepared in one example of the invention.

Detailed Description

Reference will now be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment.

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 invention relates to a preparation method of a type b haemophilus influenzae capsular polysaccharide, which comprises the following steps:

a) carrying out ultrafiltration concentration on the fermented clarified liquid to obtain a first concentrated liquid;

b) adding a nucleic acid removing liquid into the first concentrated solution, stirring, centrifuging, collecting supernatant, and performing ultrafiltration concentration to obtain a second concentrated solution;

the nucleic acid removing liquid is 30-50% (v/v) ethanol solution containing 0.001-0.003 mol/L EDTA, 4-8% (g/ml) sodium acetate and 0.5-1.5% (g/ml) sodium deoxycholate;

the volume of the first concentrated solution and the second concentrated solution is independently selected from 1/10-1/3 of the volume of the fermentation clarified solution;

c) adding CTAB into the second concentrated solution to a final concentration of 0.5-1.5% (g/ml) for incubation so as to extract capsular polysaccharide, stirring, centrifuging, collecting precipitate, and re-dissolving the precipitate with 65-75% (v/v) ethanol so as to dissolve the capsular polysaccharide to obtain a composite polysaccharide extracting solution;

d) and centrifuging the composite polysaccharide extracting solution, collecting supernatant, dissociating by using inorganic salt to remove CTAB, and then precipitating capsular polysaccharide by alcohol.

According to the invention, the fermentation clarified liquid is subjected to ultrafiltration concentration, so that a part of impurities are removed firstly, the pressure of subsequent impurity removal operation is reduced, the operation volume is reduced, and the operation convenience is improved; the method is characterized in that most of nucleic acid, protein and endotoxin are removed by adding low-concentration ethanol containing EDTA, sodium acetate and DOC with specific concentration into the fermentation concentrated solution, so that the phenol extraction step is replaced, and the operation difficulty and the operation safety risk are reduced; the method has the advantages of high yield, stable output, low impurity content and the like.

In some embodiments, the fermentation supernatant can be prepared by known methods, for example, by adding formaldehyde solution to the harvested culture medium for sterilization, centrifuging the sterilized culture medium to remove cells, and collecting the supernatant.

In some embodiments, the nucleic acid removing solution is a 35% to 45% ethanol solution containing 0.0015mol/L to 0.0025mol/L EDTA, 5% to 7% (g/ml) sodium acetate, and 0.7% to 1.3% (g/ml) sodium deoxycholate.

In some embodiments, the de-nucleating agent solution is a 40% ethanol solution containing 0.002mol/L EDTA, 6% (g/ml) sodium acetate, and 1% (g/ml) sodium deoxycholate.

In some embodiments, the CTAB is added to the second concentrate at a concentration of 8% to 12% (g/ml).

In some embodiments, the final concentration of CTAB is 0.7% to 1.3% (g/ml), and may be 1%.

In some embodiments, the volume ratio of the fermentation concentrate to the nucleic acid removal solution is 1: (1.5-2.5), and may be 1: 2.

in some embodiments, the method of dissociating to remove CTAB comprises:

adding sodium chloride into the supernatant to a final concentration of 0.4-0.6M, stirring for 30min at 2-8 ℃, centrifuging, and collecting the precipitate;

dissolving the precipitate in 30-50% (v/v) ethanol containing 0.3-0.5M sodium chloride, and centrifuging to collect the supernatant.

In some embodiments, step d) is followed by step e): redissolving the capsular polysaccharide obtained by alcohol precipitation with water, ultrafiltering and concentrating, collecting the trapped fluid and drying. The method of drying is preferably freeze drying.

In some embodiments, the ultrafiltration concentration rate membrane has a molecular weight cut-off of 90-110 k, or 95k, 105k, and most preferably 100 k.

In some embodiments, the stirring time in step b) is 2h to 4 h.

In some embodiments, the stirring time in step c) is 4 to 8 hours.

In some embodiments, the redissolving of the precipitate with 65% to 75% (v/v) ethanol is performed under stirring for 8h to 16 h.

In the present invention, unless otherwise specified, the stirring time described for incubation, reconstitution, etc. is preferably calculated at room temperature, for example, 18 ℃ to 30 ℃ or 20 ℃, 22 ℃, 25 ℃, 27 ℃; preferably, the temperature is low, for example, 2 ℃ to 8 ℃, 3 ℃, 4 ℃, 5 ℃, 6 ℃, 7 ℃.

In some embodiments, the serotype of haemophilus influenzae type b is selected from one or more of a, b, c, d, e, f.

The invention also relates to the type b haemophilus influenzae capsular polysaccharide prepared by the method.

The invention also relates to a vaccine containing the composition as described above.

The composition has high polysaccharide content, and low content of impurities such as protein, nucleic acid and endotoxin. The purity is higher and the use is safer.

As mentioned above, the method provided by the invention can be used without organic reagents such as phenols and acetone which are commonly used in the traditional capsular polysaccharide purification process, and CTAB is added into the concentrated solution, so that the dosage is less, but the extraction efficiency is higher. Meanwhile, the quality of the final product far exceeds the requirements of pharmacopoeia, the contents of protein, nucleic acid and other impurities are lower, the use is safer in the future, and the obtained product can be applied to the preparation of vaccines.

Embodiments of the present invention will be described in detail with reference to examples.

EXAMPLE 1 first batch of Haemophilus influenzae type b capsular polysaccharide preparation

1. Ultrafiltering and concentrating the fermented clear liquid

Carrying out ultrafiltration concentration on the fermentation clarified liquid by adopting a 100K membrane package, and controlling the volume of the fermentation concentrated liquid to be 1/5 of the volume of the fermentation clarified liquid;

2. nucleic acid removal

Adding the mixture into a fermentation concentrated solution according to the volume ratio of 1: 2, adding 40% ethanol solution (0.002mol/L of EDTA, 6% sodium acetate and 1%), and stirring for 3 hours at room temperature;

3. collecting the crude polysaccharide

Centrifuging, collecting supernatant, and ultrafiltering and concentrating the supernatant with 100K membrane;

4. collecting the complex polysaccharide

Adding 10% CTAB into the ultrafiltration concentrated solution to a final concentration of 1%; stirring for 6 hours at room temperature; centrifuging and collecting the precipitate; dissolving the compound polysaccharide with 70% ethanol, and stirring at room temperature for 12 h;

5. collecting the ethanol solution of the compound polysaccharide

Centrifuging and collecting supernatant;

6. collecting the salted polysaccharide

Adding 2M sodium chloride solution into the centrifugal supernatant to a final concentration of 0.5M, and stirring for 30min at 2-8 ℃; centrifuging and collecting precipitate;

7. collecting the ethanol solution

Dissolving the salt precipitated polysaccharide with 30-50% ethanol (containing 0.4M sodium chloride); centrifuging and collecting the supernatant;

8. alcohol precipitated polysaccharides

Adding ethanol to 90%, mixing, and standing overnight; centrifuging and collecting precipitate;

9. ultrafiltration

Redissolving with purified water, then carrying out ultrafiltration concentration by using a 100K membrane pack, and collecting trapped fluid, namely polysaccharide stock solution;

10. freeze-drying

Freeze drying and storing.

EXAMPLE 2 second batch Haemophilus influenzae type b capsular polysaccharide preparation

1. Ultrafiltering and concentrating the fermented clear liquid

Carrying out ultrafiltration concentration on the fermentation clarified liquid by adopting a 100K membrane package, and controlling the volume of the fermentation concentrated liquid to be 1/10 of the volume of the fermentation clarified liquid;

2. nucleic acid removal

Adding the mixture into a fermentation concentrated solution according to the volume ratio of 1: 1.4 adding 50% ethanol solution (0.001mol/L EDTA, 8% sodium acetate and 0.5%), stirring at room temperature for 3 h;

3. collecting the crude polysaccharide

Centrifuging, collecting supernatant, and ultrafiltering and concentrating the supernatant with 100K membrane;

4. collecting the complex polysaccharide

Adding 10% CTAB into the ultrafiltration concentrated solution to a final concentration of 0.5%; stirring for 6 hours at room temperature; centrifuging and collecting the precipitate; dissolving the compound polysaccharide with 65% ethanol, and stirring at room temperature for 12 h;

5. collecting the ethanol solution of the compound polysaccharide

Centrifuging and collecting supernatant;

6. collecting the salted polysaccharide

Adding 2M sodium chloride solution into the centrifugal supernatant to a final concentration of 0.6M, and stirring for 30min at 2-8 ℃; centrifuging and collecting precipitate;

7. collecting the ethanol solution

Dissolving the salt precipitated polysaccharide with 30-50% ethanol (containing 0.3M sodium chloride); centrifuging and collecting the supernatant;

8. alcohol precipitated polysaccharides

Adding ethanol to 90%, mixing, and standing overnight; centrifuging and collecting precipitate;

9. ultrafiltration

Redissolving with purified water, then carrying out ultrafiltration concentration by using a 100K membrane pack, and collecting trapped fluid, namely polysaccharide stock solution;

10. freeze-drying

Freeze drying and storing.

EXAMPLE 3 third batch Haemophilus influenzae type b capsular polysaccharide preparation

1. Ultrafiltering and concentrating the fermented clear liquid

Carrying out ultrafiltration concentration on the fermentation clarified liquid by adopting a 100K membrane package, and controlling the volume of the fermentation concentrated liquid to be 1/3 of the volume of the fermentation clarified liquid;

2. nucleic acid removal

Adding the mixture into a fermentation concentrated solution according to the volume ratio of 1: 2.5 adding 30% ethanol solution (0.003mol/L EDTA, 4% sodium acetate and 1.5%), stirring at room temperature for 3 h;

3. collecting the crude polysaccharide

Centrifuging, collecting supernatant, and ultrafiltering and concentrating the supernatant with 100K membrane;

4. collecting the complex polysaccharide

Adding 10% CTAB into the ultrafiltration concentrated solution to a final concentration of 1.5%; stirring for 6 hours at room temperature; centrifuging and collecting the precipitate; dissolving the compound polysaccharide with 75% ethanol, and stirring at room temperature for 12 h;

5. collecting the ethanol solution of the compound polysaccharide

Centrifuging and collecting supernatant;

6. collecting the salted polysaccharide

Adding 2M sodium chloride solution into the centrifugal supernatant to a final concentration of 0.4M, and stirring for 30min at 2-8 ℃; centrifuging and collecting precipitate;

7. collecting the ethanol solution

Dissolving the salt precipitated polysaccharide with 30-50% ethanol (containing 0.5M sodium chloride); centrifuging and collecting the supernatant;

8. alcohol precipitated polysaccharides

Adding ethanol to 90%, mixing, and standing overnight; centrifuging and collecting precipitate;

9. ultrafiltration

Redissolving with purified water, then carrying out ultrafiltration concentration by using a 100K membrane pack, and collecting trapped fluid, namely polysaccharide stock solution;

10. freeze-drying

Freeze drying and storing.

The haemophilus influenzae type b capsular polysaccharide prepared in examples 1-3 was assayed according to the requirements associated with the pharmacopoeia of the people's republic of China, the current edition.

According to identification results, the content of protein, nucleic acid and endotoxin in the prepared Haemophilus influenzae type b capsular polysaccharide is 0.65g/L, and the expression level of the polysaccharide is 0.65 g/L.

The nuclear magnetic resonance hydrogen spectrograms of the capsular polysaccharide prepared in the examples 1 to 3 are shown in the figures 1 to 3 in sequence.

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 shall be subject to the appended claims.