KR20010054032A - Purification method for O-specific antigen of Shigella flexneri 2a - Google Patents

Abstract

PURPOSE: A method for purifying O-specific antigen of Shigella sp. is provided, thereby the O-specific antigen of Shigella sp. having high purity can be purified without losing its antigenicity, so that vaccines for Shigella sp. can be developed. CONSTITUTION: The method for purifying O-specific antigen of Shigella sp. comprises the steps of: incubating Shigella flexneri 2A in a brain extract liquid medium containing glycerol at 37 deg. C; adding hexadecyltrimethyl ammonium bromide into the fermented culture to inactivate Shigella flexneri 2A; centrifuging the culture and collecting the supernatant; adding phenol into the supernatant to remove proteins from the antigen extract; adding acetic acid into the solution to separate Lipid A and O-specific polysaccharide from LPS; adding tris buffer solution containing 1% of EDTA and 1% of Triton X-100 into the solution to remove Lipid A; adding hydrophobic resin XAD-16HP into the solution and reacting them; removing resins and ultracentrifuging them; adding calcium chloride and ethanol into the solution, and centrifuging them to collect the precipitate; and drying the precipitate under vacuum condition.

Description

Purification method for bacterial antigen of bacterial heterozygos {Purification method for O-specific antigen of Shigella flexneri 2a}

Shegella is a gram-negative bacterium belonging to the Enterobacteriaceae family. It is a gram-negative bacterium that has no motility, no capillaries, and no apoptosis. It is divided into four species: S.flexneri, S.sonnei, S.doydii, and S.dysenteriae. Bacterial dysentery still occurs in many parts of the world. It is highly infectious and can be transmitted only by bacteria in the hands of patients or carriers without proliferation in the media, and mucosal infection is the main method of spreading Shigella infections every year. People are suffering from the disease, and 650,000 of them are dead.

The incidence of bacterial dysentery has been decreasing in Korea. Since 1998, the outbreak has increased dramatically, such as group outbreaks nationwide. In addition, the heterologous bacteria isolated after 1998 have shown a serious level of resistance to antibiotics, such as resistance to antibiotics such as tetracycline and streptomycin.

Although the immune mechanism against Shigella is still unclear, there are studies showing that O-specific antigen acts as a protective antigen. Not. Vaccines are being developed into synthetic strains of Shigella and Escherichia coli, attenuated live vaccines, component vaccines based on LPS and polysaccharides, and conjugated vaccines for the epic polysaccharide antigens and proteins. It's going on. In the field of conjugation vaccines of ACE polysaccharide antigens and proteins, they are developed by IDF in Israel and NIH in the United States, and have been reported to have 74% protection against Shigella in Israel.

Therefore, the present invention is a technique of the invention in the development of a pure separation and purification method of the Osteopolysaccharide antigen, which is one of the key points in the development of vaccines against bacterial dysentery.

It is an object of the present invention to promote the development of a prophylactic vaccine against Shigella by purely purifying the Shigella bacteria O-specific polysaccharide antigen (O-specific polysaccharide antigen) to a high level of immunogenicity without damage.

In the present invention, Shigella strains (S. flexneri 2A) are used as Shigella bacteria for antigen extraction and Brain Heart infusion broth is used as a medium for strain culture. In order to achieve the above object, the step of culturing Shigella strains and inactivating the culture strain, extracting and purifying the O-specific polysaccharide antigen, and finally drying the purified purified antigen It provides a method for pure purification of O-specific polysaccharide antigen (O-specific polysaccharide antigen) comprising the step of powdering.

Example 1 Culture of Shigella Strains

Shigella strains (Shigella flexneri 2A) were incubated for 12 hours at 35 ° C. in brain leachate liquid medium (Difco0037-01-6) to which 30% glycerol was added, and then passaged to Mekongki plate medium (Difco 0075-17-1). The colonies are taken and cultured again in the brain leaching medium. Culture conditions in the brain leaching medium was shaken for 6 hours at 35 ℃ and then transferred to a new brain acupuncture medium and cultured at 35 ℃ for 16 hours. 10% hexadecyltrimethylammonium bromide (Sigma H-5882) solution was added to the culture to make the final concentration to 1% and stirred for 30 minutes to inactivate the bacteria.

Example 2 Extractive Separation and Purification of O-Specific Polysaccharide Antigen

Centrifuged (10500g) the inactivated culture solution to remove the supernatant, and the precipitate is suspended in 1M calcium solution and homogenized. The bacterial suspension is stirred for 40 minutes at 350 rpm using an impeller stirrer to extract the antigen of the bacteria. Add 99% ethanol to the antigen extract so that the final concentration is 25v / v%, homogenize by further stirring for 10 minutes, centrifuged for 30 minutes with a centrifugal force of 20000g to recover the supernatant and discard the precipitate. Add 99% ethanol to the centrifugal supernatant again to reach a final concentration of 80%, stir for 30 minutes, and centrifuge for 30 minutes with a centrifugal force of 10500 g to recover the antigen. The recovered antigen is dissolved in 0.5 M sodium acetate solution, and then phenol solution is added and mixed for at least 30 minutes. Centrifuge at 15000 rpm for 30 minutes to remove the upper aqueous layer. After adding the phenol solution to the aqueous layer again, the mixture was stirred for 60 minutes, centrifuged at 15000 rpm for 30 minutes, and the upper aqueous solution layer was recovered to remove the protein from the antigen extract. The recovered aqueous solution was dialyzed in distilled water, and then acetic acid was added to a concentration of 2% acetic acid with stirring, followed by reaction for 2 hours to separate Lipid A and O-specific polysaccharide (O-specific polysaccharide) from LPS. . After separation, 1M calcium solution was added to the antigen solution to make 0.2% concentration, and then 99% ethanol was added to make 80% concentration, and the precipitate was recovered by centrifugation at 10500 rpm. To remove Lipid A from the recovered precipitate, dissolve the precipitate in physiological saline and adjust the concentration to 4 mg / mL, which contains 2% EDTA with Chilean agent and 2% Triton X-100 as Detergent. 100 mM Tris buffer was added (final 1% EDTA, 1% Triton X-100). The pH range of Tris buffer was 8.1-8.2, and hydrophobic resin, XAD-16HP, was added at a ratio of 50 grams per gram of antigen to the mixed solution of buffer and antigen and reacted for 3 hours. After the reaction, the resin is removed from the solution, and the solution is ultrafiltration to remove all the components of the buffer. After the ultrafiltration solution was added to 1M Kalshu chloride solution was adjusted to the final 0.1M concentration, ethanol was added to precipitate the polysaccharide antigen and the ethanol treatment solution was centrifuged at 10500g to recover the precipitate. Recovery of O-specific polysaccharides from lipopolysaccharide (LPS), removal rate of Lipid A was confirmed by LAL test, and the level of LAL before hydrophobic resin treatment was 100%. After the resin treatment, about 95% of the lipid a was removed.

Example 3 Drying of Separated Polysaccharide Antigens

The precipitate recovered by ethanol treatment was suspended in 99% pure ethanol. After homogenizing the antigens suspended in pure ethanol, the filter was filtered to collect only the antigens, which were then suspended in pure acetone and homogenized. The acetone floating antigen was again filtered through filter paper to collect only the antigen, and then the acetone was removed. Then, the antigen was put in a vacuum container, and the vacuum was evaporated to remove the acetone.

Example 1 Effect of Triton X-100 Concentration in Separation of O-Specific Polysaccharides from Lipid A through Lipid A through Examples 1, 2 and 3 The change of LAL value according to the EDTA concentration change in% was performed as follows. The initial LAL concentration was 327 endotoxin units per microgram of polysaccharide.

Final EDTA Concentration EU / ug of polysaccharide Polysaccharide Recovery (%) One% 19 96 2% 15 91 3% 12 85

Experimental Example 2 Experimental experiments were carried out on the change of the LAL value with respect to the EDTA concentration of 1% and the Triton X-100 concentration change, and the initial LAL concentration was polysaccharide. 327 endotoxin units (EU) per microgram.

Final Triton concentration EU / ug of polysaccharide Polysaccharide Recovery (%) One% 19 96 2% 6 82 3% 2 81

The effective concentrations of Triton X-100 and EDTA to remove lipid a through the effect tests example 1 and 2 can be increased and the lipid concentration can be removed when treated with a concentration of 1%. You can see that.

The present invention is to develop a method for separating and purifying O-specific polysaccharide (O-specific polysaccharide) from the lipopolysaccharide (LPS) of Shigella and to facilitate the development of vaccines against Shigella bacteria It works.

Claims (5)

Separation and purification of O-specific polysaccharide (O-specific polysaccharide) from Shigella lipopolysaccharide (LPS) Method of using lipopolysaccharide of S. flexneri 2A according to claim 1 for lipopolysaccharide The method of claim 1 using XAD 16HP for hydrophobic resins. Method of using Triton X 100 as a detergent according to claim 1 How to Use EDTA as a Chileanizing Agent