WO2017036140A1

WO2017036140A1 - A-group neisseria meningitidis cultivation method

Info

Publication number: WO2017036140A1
Application number: PCT/CN2016/078086
Authority: WO
Inventors: 朱冲; 米强; 陈道远
Original assignee: 成都欧林生物科技股份有限公司
Priority date: 2015-08-31
Filing date: 2016-03-31
Publication date: 2017-03-09
Also published as: CN105039227A

Abstract

An A-group Neisseria meningitidis cultivation method comprises the following steps: (1) preparing purified strains of a working seed lot; (2) opening the strains of the working seed lot, inoculating same into a semi-synthetic culture medium, performing cultivation at the temperature of 35-37°C for 16-24 hours, and then preparing an appropriate quantity of seeds for production after three-generation multiplication culture; and (3) inoculating the seeds for production into a fermentation tank, and performing cultivation for 6-12 hours, the culture medium in the fermentation tank being a semi-synthetic culture medium, the temperature being 35-37°C, the pH of the culture medium being 7.0-7.2, the stirring speed in the fermentation tank being 140-150 r/min, and the inoculation quantity being 100,000 ml to 150,000 ml.

Description

Specification Name of Invention: Method for culturing group A meningococcal bacteria

[0001] The present invention relates to a method for cultivating a strain, and particularly relates to a method for culturing a group A meningococcus.

Background technique

[0002] Epidemic cerebrospinal meningitis (abbreviated as ECM, the same below) is an acute respiratory infection caused by Neisseria mening gitidis. For more than 100 years, it has been prevalent or scattered around the world, causing sepsis and meningitis after infection with pathogens. The susceptible population is mainly children, with the highest rate of fulminant deaths, up to 40<3⁄4~60<3⁄4. The incidence rate in every continent in the world is between 1/100,000 and 10/10 million, and the total case fatality rate is 5%~15<3⁄4. Up to 20% of meningitis patients have neurological sequelae, including mental impairment and deafness. Serological classification according to the capsular polysaccharide type can be divided into 13 serotypes, of which group A, group B, and group C account for about 90% of the epidemic group. Group A meningococcal is a major epidemic of the major pathogenic serogroups, especially in the so-called African “Brain Brain Epidemic Belt”, which is a major epidemic every 7-14 years.

[0003] There were five national epidemic epidemics in China in 1938, 1949, 1959, 1967, and 1977; among them, the most prevalent in the spring of 1967, the incidence rate was as high as 403/100,000, and the mortality rate was 5.49<3⁄4. In the past 90% of cases in China, the disease of group A was caused by disease. Now, group B or group C germs have convulsions. Since the beginning of 2003, the incidence of C group cerebral palsy has increased significantly. At present, group A and group C account for more than 50% of all serogroups, and group C still has a tendency to increase further. Therefore, the focus of prevention of epidemic work in China is to prevent group A and group C.

[0004] The most effective way to prevent epidemic cerebrospinal meningitis is to vaccinate. Studies have shown that the capsular polysaccharide of group A group C meningococcus has good immunogenicity, and the capsular polysaccharide can be directly prepared into a vaccine, and the immunized population can obtain immune protection. Since 2007, China has included meningococcal vaccines into national immunization programs, and has universally vaccinated suitable children across the country. The meningococcal polysaccharide vaccine currently included in the national immunization program includes group A meningococcal polysaccharide vaccine, group A group C meningococcal polysaccharide vaccine.

[0005] In the prior art, polysaccharides are extracted from group A meningococcus to form a polysaccharide vaccine, thereby achieving the effect of preventing meningitis. Because the polysaccharide is extracted from the capsule, if there are more dead bacteria in the culture process of the strain, it is easy to cause the extraction efficiency of the polysaccharide extracted directly from the group A meningococcus is low, and the extracted polysaccharide is not Easy to purify.

technical problem

[0006] The object of the present invention is to solve the problem that the extraction efficiency of the polysaccharide extracted directly from group A meningococcus is low in the prior art, and the extracted polysaccharide is not easy to be purified, and a group A meningitis which solves the above problems is provided. Method of cultivating cocci.

Problem solution

Technical solution

[0007] In order to solve the above disadvantages, the technical solution of the present invention is as follows:

[0008] A method for culturing group A meningococcus includes the following steps:

[0009] (1) preparing a purified working seed strain;

[0010] (2) 工作工作 working seed batch strain, inoculated into semi-integrated medium, cultured at a temperature of 35~37 ° C for 16~24 吋, and then three-generation expansion culture to prepare a suitable amount of production Seed

[0011] (3) The seed for production is inoculated into the fermenter for 6 to 12 hours; the medium in the fermenter is a semi-integrated medium, the temperature is 35 to 37 ° C, and the pH of the medium is 7.0 to 7.2. The stirring speed in the fermenter was 140 to 1 50 r/min, and the inoculum amount was 10 to 150,000/ml.

[0012] Preferably, the specific process of the step (1) is as follows:

[0013] A group of N. meningitidis strains were inoculated on a purification medium, and incubated at 38 ° C for 20 h, and the robust and no-bacteria-contaminated strains were selected and then inoculated onto the purification medium for secondary treatment. The culture was incubated at 38 ° C for 12 h, and the strains with strong and no fungus contamination were selected and added to the sterile skim milk, and the purified working seed strains were prepared by mixing and lyophilizing.

[0014] As a preferred embodiment, the process of purifying the purified seed is as follows:

[0015] Dissolving the working seed strain in sterile water, inoculation on 10% sheep blood agar medium, and placing

After culturing for 16 to 20 hours in an environment of 35 to 37 ° C, select a strain that is robust and free of contamination by bacteria.

[0016] Further, the specific cultivation process of the step (2) is as follows:

[0017] The purified seed after inoculation is inoculated into a semi-integrated liquid medium for activation culture; the conditions of the activation culture are temperature 35 to 37 ° C, pH 7.0 to 7.2, stirring speed 140 to 150 r / min, culture 吋The interval is 6~8h; finally, the activated purified seeds are cultured in three generations to prepare a suitable number of production seeds. [0018] Further, the conditions for the three-generation expansion culture are: the culture medium for expansion culture is a semi-integrated liquid medium; the incubation conditions are temperature 35 to 37 ° C, pH 7.0 to 7.2, stirring speed 140 ~150r/min.

[0019] In order to achieve the best results, the composition of the semi-integrated liquid medium is as follows:

[0020] Beef leachate 1L, peptone 10g, sodium chloride 5g, sterile defibrinated horse blood, sheep blood or rabbit blood 100ml, glucose 3g, hydrochloric acid casein hydrolysate 2g, pH 7.0~7.2.

Advantageous effects of the invention

Beneficial effect

[0021] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0022] The group A meningococcal bacteria cultivated by the invention have fewer dead bacteria, and the extracted polysaccharide is more easily purified; and the extraction rate of the polysaccharide in the capsule is higher.

Embodiments of the invention

The present invention will be further described in detail below with reference to the embodiments, but the embodiments of the present invention are not limited thereto.

Embodiment 1

[0025] A method for culturing group A meningococcus comprises the following steps:

[1] (1) Preparation of purified working seed strains: Inoculating Group A N. meningitidis strains onto a purification medium, culturing at 38 ° C for 20 hours, selecting robust and non-organic contamination The strain is inoculated on the purification medium for secondary culture, and then incubated at 38 ° C for 12 h. The robust and bacteria-free bacteria are added to the sterile skim milk, and the mixture is lyophilized to obtain purified. Working seed strains.

[0027] (2) Dissolving the working seed strain in sterile water, inoculation on 10% sheep blood agar medium, and culturing in the environment of 35~37 ° C for 16~20 hours, then selecting a robust and no-bacteria-contaminated strain;

[0028] The purified seed after inoculation is inoculated into a semi-integrated liquid medium for activation culture; the conditions of the activation culture are temperature 35 to 37 ° C, pH 7.0 to 7.2, stirring speed 140 to 150 r/min, and culture 吋The interval is 6~8h;

[0029] Finally, the activated purified seed is subjected to three generations of expansion culture to prepare a suitable amount of production seeds; wherein the medium for expansion culture is semi-integrated liquid medium; the cultivation condition is temperature 35 to 37 ° C , p H7.0~7.2, stirring speed 140~150r/min.

[0030] (3) inoculating the production seed into the fermenter for 6 to 12 hours; the medium in the fermenter is semi-complex The medium is mixed at a temperature of 35 to 37 ° C, the pH of the medium is 7.0 to 7.2, the stirring speed in the fermenter is 140 to 1 50 r/min, and the inoculum is 10 to 150,000/ml.

[0031] In this embodiment, the specific cultivation conditions in each step are as follows:

[0032] Step (2) The working condition of the seed strain is 35 ° C, and the culture is carried out for 18 hours; the conditions of the activation culture are the temperature of 37 ° C, the pH of 7.2, the stirring speed of 140 r / min, and the cultivation of the daytime. 6~8h; the incubation conditions for the expansion culture are temperature 36 ° C, pH 7.0, stirring speed 145r / min;

[0033] The fermentation condition of the fermenter culture in the step (3) is a temperature of 37 ° C, a pH of the medium of 7.0, a stirring speed of 145 r / min, and an inoculum amount of 120,000 / ml.

[0034] and the purification medium used in the step (1) is a chocolate medium added with an antibiotic, which is effective for the purpose of purification; the medium used in the step (2) of the seed seed strain is the chocolate without antibiotic added. The medium, that is, 10% sheep blood agar medium, can effectively achieve the effect of the seed strain of the seed by the culture of the medium.

[0035] The medium used for the culture for activation and amplification is a semi-integrated liquid medium, and the ratio of the medium is: beef leachate 1 L, peptone 10 g, sodium chloride 5 g, sterile defibrinated horse blood, sheep blood Or rabbit blood 100ml, glucose 3g, hydrochloric acid casein hydrolysate 2g, pH 7.0~7.2.

Embodiment 2

[0037] The difference between this embodiment and Embodiment 1 is only that the specific cultivation conditions in each step are different, and the specific settings are as follows:

[0038] Step (2) The working seed culture strain is cultured at 36 ° C, and cultured for 16 hours; the conditions of activation culture are temperature 37 ° C, pH 7.0, stirring speed 145 r / min, culture daytime 8h; the cultivation conditions for the amplification culture are temperature 37 ° C, pH 7.0, stirring speed 150 r / min;

[0039] The culture condition of the fermenter culture in the step (3) is a temperature of 37 ° C, a pH of the medium of 7.0, a stirring speed of 150 r / min, and an inoculum amount of 150,000 / ml.

Embodiment 3

[0041] The difference between this embodiment and Embodiment 1 is only that the specific cultivation conditions in each step are different, and the specific settings are as follows:

[0042] Step (2) The working condition of the seed strain is 37 ° C, and the culture is carried out for 18 hours; the conditions of the activation culture are the temperature of 36 ° C, the pH of 7.2, the stirring speed of 150 r / min, and the cultivation of the daytime. 7h; cultivation of expansion culture The conditions are temperature 37 ° C, pH 7.2, stirring speed 145 r / min;

[0043] The fermentation condition of the fermenter culture in the step (3) is a temperature of 37 ° C, a pH of the medium of 7.2, a stirring speed of 145 r / min, and an inoculum amount of 100,000 / ml.

The above embodiments are merely preferred embodiments of the present invention, and are not intended to limit the scope of the present invention, and any changes made by the design principles of the present invention and non-inventive work on the basis of the present invention belong to the present invention. Within the scope of protection.

Claims

Claim

[Claim 1] A method for culturing a group A meningococcal strain, comprising the steps of:

(1) preparing a purified working seed strain;

(2) 工作工作 working seed batch strain, inoculated into semi-integrated medium, cultured at a temperature of 35~37 °C for 16~24 hours, and then subjected to three generations of expansion culture to prepare a suitable amount of production seeds;

(3) Inoculate the production seeds into the fermenter for 6~12 hours; the medium in the fermenter is semi-integrated medium, the temperature is 35~37°C, the medium pH is 7.0~7.2, in the fermenter The stirring speed was 140 to 150 r/min, and the inoculum amount was 10 to 150,000/ml.

The method for culturing a group A meningococcus according to claim 1, wherein the specific process of the step (1) is as follows:

A group of N. meningitidis strains were inoculated on the purification medium, and incubated at 38 ° C for 20 h. The robust and no-bacteria-contaminated strains were selected and then inoculated onto the purification medium for secondary culture. After incubating at 38 °C for 12 h, select the robust and bacteria-free bacteria into the sterile skim milk, and mix and freeze to prepare the purified working seed strain.

The method for culturing group A meningococcus according to claim 1, wherein the process of purifying the seed is as follows:

The working seed batch is dissolved in sterile water, inoculated on 10% sheep blood agar medium, and cultured in the environment of 35~37 ° C for 16~20 hours, and then selected to be robust and free of bacteria. Contaminated bacteria can be used.

The method for culturing group A meningococcus according to claim 3, wherein the specific cultivation process of the step (2) is as follows:

The purified seeds after inoculation are inoculated into a semi-integrated liquid medium for activation culture; the conditions for activation culture are temperature 35 to 37 ° C, pH 7.0 to 7.2, stirring speed 140 to 150 r/min, and culture time is 6 ~8h; Finally, the activated purified seeds are subjected to three generations of expansion culture to prepare a suitable amount of production seeds.

The method for culturing group A meningococcal bacteria according to claim 4, wherein the conditions for the three-generation expansion culture are: the medium for expansion culture is a semi-integrated liquid medium Medium; incubation conditions are temperature 35~37 ° C, pH 7.0~7.2, stirring speed 140~150r/min

The method for culturing group A meningococcal bacteria according to claim 1, wherein the composition of the semi-integrated liquid medium is as follows:

Beef leachate 1L, peptone 10g, sodium chloride 5g, sterile defibrinated horse blood, sheep blood or rabbit blood 100ml, glucose 3g, hydrochloric acid casein hydrolysate 2g, pH 7.0~7.2.