황색포도상구균 독소 B 분리 방법에 대한 연구

Abstract

경제 발전에 따른 소득수준의 향상으로 국민의 외식 및 가공식품에 대한 의존도가 증가하였으며 더불어 식품 안전에 대한 관리기준이 강화되고 있다. 그럼에도 식중독, 유해한 화학물질, 이물질 유입 사고 등 안전을 위협하는 사고는 끊임없이 나타나고 있는데 이 중 식중독과 같은 생물학적 위해요소로 인한 사고는 예측하기 어려울 뿐만 아니라 한 번에 다수의 피해자가 발생하므로 철저한 위생⋅안전 관리가 요구된다. 식중독을 발생시키는 원인 중 하나인 황색포도상구균은 사람이나 동물에 상재해 있고, 식품 작업 환경에 의해 오염되기 쉽기 때문에 연중 꾸준히 발생하여 우리나라뿐만 아니라 전 세계적으로 문제시 되는 주요 식중독 원인균이다. 황색포도상구균 식중독은 황색포도상구균이 산출하는 장독소에 의해 발생하며 원인균으로부터 오염, 오염된 식품에서의 원인균의 증식 및 장독소 생산의 3단계를 거쳐 발현된다. 16가지의 황색포도상구균 장독소 중 B형(Staphylococcal enterotoxin B, SEB)은 분자량이 가장 작으며 내열성이 가장 강하고 대부분의 유통 조건에서 안정하기 때문에 독소발현 제어에 각별한 주의가 요구된다. 특히 흡입 중독을 일으키는 특성이 있어 연무제 형태의 생물학적 무기로 사용될 위험이 큰 독소이다. 인체에 연무제 노출로 인한 반수 유효선량(A half-effective dose, ED50)은 0.0004 ㎍/kg bw이며 반수 치사량(A half-lethal dose, LD50)은 0.02 ㎍/kg bw이다. 단순 단백질인 황색포도상구균 장독소 B형을 균체 및 독소가 산출된 배지로부터 분리하는 방법은 조분획, 크로마토그래피, 전기영동법 등이 있으며 본 연구에서는 단백질을 변성시키지 않고 분리할 수 있는 액체 크로마토그래피를 이용하였다. 액체 크로마토그래피 중 단백질을 신속하게 분리할 수 있는 Fast Protein Liquid Chromatography(FPLC) 기법을 이용하였으며, Ion exchange chromatography와 Gel filtration chromatography의 두 단계를 거쳐 분리⋅정제하였다. 연구 결과, Cation exchange chromatography에 적합한 레진과 버퍼의 pH는 WorkBeads 40S(pH 7.5)이었으며 Gel filtration chromatography에 적합한 레진은 Sephacryl S-100에서 최적의 분리능을 보였다. 또한 시료의 전기전도도를 낮추고 농축을 기대하기 위하여 사용한 투석 및 버퍼 치환 방법은 단백질의 손실을 가져오는 것으로 판단되어 이는 효과적인 방법이 아니었다. 이를 대체하기 위한 방안으로 5 mM Sodium phosphate 버퍼(이온 강도 0.02 M, pH 7.5)로 10배 희석한 시료를 사용하였다. 분리한 독소의 농도를 확인하기 위하여 표준검량곡선에 시료의 흡광도를 대입하였으며 약 100 ㎍/mL의 독소를 분리 및 정제한 것으로 확인되었다. 따라서 본 연구는 황색포도상구균 장독소 분리 및 정제 방법에 대한 기초적인 조건 확립에 대한 연구였으며, 향후 기타 장독소 분리에 응용할 수 있고 식중독 독소의 신속 진단법 개발 및 항체 생산 발전 연구에 활용하여 국민의 식생활 안전에 기여하고자 한다.| Management guidelines of food safety were tighten up along with increased eat-out habits and consumption of processed foods. But foodborne outbreaks related to pathogens, harmful chemical substance, and foreign materials still occur. Among them, food poisoning incidents caused by microbiological hazards are most problematic because they are unpredictable and many people can get sick. Staphylococcus aureus is one of the serious causes of food poisoning. It is a common bacterium found on the skin and in the noses of healthy people and animals. Food poisoning from S.aureus is occured from Staphylococcal Enterotoxins(SEs). S.aureus has the ability to produce 16 different toxins. Among toxins, Staphylococcal Enterotoxin B(SEB) is the smallest, the strongest to heat-resistance, and stable in most of marketing conditions. Especially SEB can causing inhalation poisoning, so it can be a bio-terrorism agent and needs a special attention. Many studies reported that a half-effective dose(ED50) of SEB is 0.0004 ㎍/kg bw and a half-lethal dose(LD50) is 0.02 ㎍/kg bw in human for aerosol exposure. This study used liquid chromatography for separation of SEB from S.aureus bacteria and incubated medium. Chromatography is one of the stable separate method that do not induce denaturalization of proteins. And fast protein liquid chromatography(FPLC) is the rapid protein separation method. In this study, ion exchange chromatography and gel filtration chromatography was applied to separate and purify SEB. Results showed that the optimized resin and buffer pH for cation exchange chromatography was WorkBeads 40S and sodium phosphate buffer(pH 7.5). Sephacryl S-100 was the optimum separation resin for gel filtration chromatography. Dialysis and buffer exchange those were performed to lower electric conductivity and concentrate SEB was unsuccessfull because of losing protein. Therefore, this step was not considered for remaining experiments. Consequently of upper experiment steps, concentration of separated and purified SEB was 100 ㎍/mL to compared with calibration curve of SEB. In conclusion, this study provided that the basic condition for separation and purification of SEB. This method can be applied to separate other enterotoxins to develop rapid detection methods for food safety.; Management guidelines of food safety were tighten up along with increased eat-out habits and consumption of processed foods. But foodborne outbreaks related to pathogens, harmful chemical substance, and foreign materials still occur. Among them, food poisoning incidents caused by microbiological hazards are most problematic because they are unpredictable and many people can get sick. Staphylococcus aureus is one of the serious causes of food poisoning. It is a common bacterium found on the skin and in the noses of healthy people and animals. Food poisoning from S.aureus is occured from Staphylococcal Enterotoxins(SEs). S.aureus has the ability to produce 16 different toxins. Among toxins, Staphylococcal Enterotoxin B(SEB) is the smallest, the strongest to heat-resistance, and stable in most of marketing conditions. Especially SEB can causing inhalation poisoning, so it can be a bio-terrorism agent and needs a special attention. Many studies reported that a half-effective dose(ED50) of SEB is 0.0004 ㎍/kg bw and a half-lethal dose(LD50) is 0.02 ㎍/kg bw in human for aerosol exposure. This study used liquid chromatography for separation of SEB from S.aureus bacteria and incubated medium. Chromatography is one of the stable separate method that do not induce denaturalization of proteins. And fast protein liquid chromatography(FPLC) is the rapid protein separation method. In this study, ion exchange chromatography and gel filtration chromatography was applied to separate and purify SEB. Results showed that the optimized resin and buffer pH for cation exchange chromatography was WorkBeads 40S and sodium phosphate buffer(pH 7.5). Sephacryl S-100 was the optimum separation resin for gel filtration chromatography. Dialysis and buffer exchange those were performed to lower electric conductivity and concentrate SEB was unsuccessfull because of losing protein. Therefore, this step was not considered for remaining experiments. Consequently of upper experiment steps, concentration of separated and purified SEB was 100 ㎍/mL to compared with calibration curve of SEB. In conclusion, this study provided that the basic condition for separation and purification of SEB. This method can be applied to separate other enterotoxins to develop rapid detection methods for food safety.