Shigella multivalent conjugate vaccine

Abstract

The invention discloses a Shigella multivalent conjugate vaccine which includes five serotypes, i.e., Shigella dysenteriae I type, Shigella flexneri 2a type, Shigella flexneri 3a type, Shigella flexneri 6 type and Shigella sonnei; the Shigella multivalent conjugate vaccine is prepared by the steps of preparing conjugate stock solutions from specific polysaccharides of the bacteria and carrier protein, and mixing five conjugate stock solutions in a proper proportion. The prepared conjugate vaccine disclosed by the invention is applicable to immunoprophylaxis of Shigella infection.

Description

1. Technical field

[0001] The invention relates to a polyvalent Shigella polysaccharide-protein conjugated vaccine preparation, especially containing Shigella flexneri type Ⅰ, Shigella flexneri type 2a, Shigella flexneri type 3a, and Shigella flexneri type 6 , Shigella sonnei 5 serotypes of Shigella multivalent conjugate vaccine. 2. Background technology

[0002] Shigella is the pathogenic bacterium that causes bacillary dysentery in humans. It is a common and frequently-occurring disease. It affects 160 million patients worldwide and causes 1.1 million deaths every year. About 80% of the cases occur in Asia. my country is an area with a relatively serious incidence of bacillary dysentery. According to the statistics of epidemic monitoring data, the incidence of bacillary dysentery ranks fourth among the statutory A and B infectious diseases, with more than 200,000 cases per year. Due to the limitation of detection methods and serious underreporting, the actual number of ca...

Examples

Embodiment 1

[0049] Fermentation of Shigella

[0050] Open the Shigella species, inoculate in 2 soytone slant, culture at 35-37°C for 12-24 hours, expand in two 500ml liquid medium, culture at 35-37°C on a shaker for 6-12 hours, Expand the seeds in 10-20L liquid medium, culture on a shaker at 35-37°C for 4-8 hours, inoculate in a fermenter containing 300-500L liquid medium, and cultivate at 35-37°C until the growth reaches the end of logarithmic growth or Stable period, during which the pH was maintained at neutral, Gram staining microscopic examination and pure bacteria experiment. The pollution-free culture is sterilized by adding formaldehyde with a final concentration of 0.5-2%, and the bacteria are separated by a continuous flow centrifuge.

Embodiment 2

[0052] lipopolysaccharide extraction

[0053] Use purified water to suspend the bacteria into a 10% bacterial suspension, add 1:1 to 95% phenol aqueous solution, stir thoroughly at 68°C for 1 hour, centrifuge, separate and harvest the water phase, and add water to the phenol phase to the original volume, Extract at 68°C for 30 minutes, centrifuge, and separate the aqueous phase. Combine the two aqueous phases, add ethanol to a concentration of 25%, supplement some salt ions, centrifuge, and remove the precipitate. Add ethanol to the supernatant to a concentration of 75%, and centrifuge to collect the precipitate. After washing with absolute ethanol and acetone, drain.

Embodiment 3

[0055] O-specific polysaccharide extraction

[0056] Dissolve lipopolysaccharide at a concentration of 1% in 1% glacial acetic acid solution, bathe in boiling water for 60-90 minutes, centrifuge at 60,000-80,000 g for 4 hours, collect the supernatant, freeze-dry, dissolve with purified water, and collect the supernatant by centrifugation. The centrifuged supernatant was purified water as the mobile phase, and was subjected to Sephadex G-25 gel chromatography to collect V 0 The nearby polysaccharide fraction, after lyophilization, is the O-specific polysaccharide.

1. Technical field

[0001] The invention relates to a polyvalent Shigella polysaccharide-protein conjugated vaccine preparation, especially containing Shigella flexneri type Ⅰ, Shigella flexneri type 2a, Shigella flexneri type 3a, and Shigella flexneri type 6 , Shigella sonnei 5 serotypes of Shigella multivalent conjugate vaccine. 2. Background technology

[0002] Shigella is the pathogenic bacterium that causes bacillary dysentery in humans. It is a common and frequently-occurring disease. It affects 160 million patients worldwide and causes 1.1 million deaths every year. About 80% of the cases occur in Asia. my country is an area with a relatively serious incidence of bacillary dysentery. According to the statistics of epidemic monitoring data, the incidence of bacillary dysentery ranks fourth among the statutory A and B infectious diseases, with more than 200,000 cases per year. Due to the limitation of detection methods and serious underreporting, the actual number of ca...

Examples

Embodiment 1

[0049] Fermentation of Shigella

[0050] Open the Shigella species, inoculate in 2 soytone slant, culture at 35-37°C for 12-24 hours, expand in two 500ml liquid medium, culture at 35-37°C on a shaker for 6-12 hours, Expand the seeds in 10-20L liquid medium, culture on a shaker at 35-37°C for 4-8 hours, inoculate in a fermenter containing 300-500L liquid medium, and cultivate at 35-37°C until the growth reaches the end of logarithmic growth or Stable period, during which the pH was maintained at neutral, Gram staining microscopic examination and pure bacteria experiment. The pollution-free culture is sterilized by adding formaldehyde with a final concentration of 0.5-2%, and the bacteria are separated by a continuous flow centrifuge.

Embodiment 2

[0052] lipopolysaccharide extraction

[0053] Use purified water to suspend the bacteria into a 10% bacterial suspension, add 1:1 to 95% phenol aqueous solution, stir thoroughly at 68°C for 1 hour, centrifuge, separate and harvest the water phase, and add water to the phenol phase to the original volume, Extract at 68°C for 30 minutes, centrifuge, and separate the aqueous phase. Combine the two aqueous phases, add ethanol to a concentration of 25%, supplement some salt ions, centrifuge, and remove the precipitate. Add ethanol to the supernatant to a concentration of 75%, and centrifuge to collect the precipitate. After washing with absolute ethanol and acetone, drain.

Embodiment 3

[0055] O-specific polysaccharide extraction

[0056] Dissolve lipopolysaccharide at a concentration of 1% in 1% glacial acetic acid solution, bathe in boiling water for 60-90 minutes, centrifuge at 60,000-80,000 g for 4 hours, collect the supernatant, freeze-dry, dissolve with purified water, and collect the supernatant by centrifugation. The centrifuged supernatant was purified water as the mobile phase, and was subjected to Sephadex G-25 gel chromatography to collect V 0 The nearby polysaccharide fraction, after lyophilization, is the O-specific polysaccharide.

Images