In this study, PAT protein of genetically modified maize was prepared from the recombinant E. coli strain BL21 (DE3), and mice were immunized with the recombinant PAT protein. After cell fusion and cloning, two hybridoma cells (PATmAb-7 and PATmAb-12) were chosen since the monoclonal antibodies (Mabs) produced by them were confirmed to be specific to PAT protein in the indirect enzyme-linked immunsorbent assay (ELISA) and western blot tests. There were no cross-reactions of either Mabs to other GM proteins or to the extracts of non-GM maize. The ELISA based on the PATmAb-7 can sensitively detect 0.3 ng/g PAT protein in corn. These results indicate that the developed Mabs can be used as bio-receptors in the development of immunosensors and biosensors for the rapid and simple detection of GM corn adulterated in foods.

인간성장호르몬(human growth hormone; hGH)은 뇌하수체 전엽에서 분비되는 호르몬 의 일종으로 단백질 합성을 촉진하고 에너지를 생산해 지방을 분해하며 뼈를 포함한 체 내의 거의 모든 조직의 성장을 자극한다. DNA 재조합 기술이 발달함에 따라 hGH는 신 체의 성장, 골밀도 향상, 체지방 감소, 세포의 재생, 근육량 증가 등에 효과를 보이는 의 약품으로 사용되고 있다. 본 연구에서는 시료 중 함유된 hGH의 효율적인 분리,정제를 위 하여 immunoaffinity chromatography system을 개발하여 hGH를 분리정제하고 그 활성도 를 측정하고자 하였다. 이를 위하여, 단크론성 항체를 생산하는 hybridoma cell line중 hGH에 가장 강한 친화력이 있는 항체를 분비하는 hGH-K-24 세포주를 선별하여 Balb/C 마우스의 복강에 주입하여 복수를 생산, 채취하였고, Protein G를 이용하여 항체를 분리 정제 하였다. 정제한 항체를 CNBr-Sepharose 4B와 결합시켜 immunoaffinity chromatography column을 제작하였고. hGH를 분비,생성하는 CHO세포주를 serum이 없는 상태에 서 10일 동안 배양한 다음 배지를 회수하여 hGH를 immunoaffinity chromatography system으로 정제하였으며, 순수정제도를 높이기 위하여 superdex G-200으로 gel filtration chromatography 하여 target 단백질인 hGH만을 분리하는 일련의 과정을 확립하였다. 또 한, hGH의 역가를 알아보기 위하여 NB2-11 cell을 이용하여 MTT assay를 통한 활성도 를 살펴보았다.

Porcine beta casein promoter를 이용하여 형질전환 동물에서 유선특이적으로 목적 단 백질을 발현시킬 수 있는 pPBC 벡터를 구축하였으며 이 벡터를 이용하여 보다 높은 농 도의 목적 단백질을 발현 시킬 수 있도록 pPBC 벡터 개량을 시도하였다. pPBC 벡터의 5'arm 부위를 5428 bp, 4419 bp, 3378 bp의 길이로 잘라 서로 다른 크기의 5’arm을 갖 게 하였으며 5’arm의 5’ 쪽으로 CMV enhancer를 삽입하였다. 또한, porcine beta casein promoter 조절 하에 hGH(human growth hormone) 유전자를 발현하도록 하는 벡터를 구 축한 후 세포주 및 형질전환 마우스에서 발현 양상을 확인하였다. 개량된 pPBC 벡터를 이용하여 mouse mammary gland epithelial cell line HC11에서 luciferase assay를 통해 각각의 벡터에 대한 활성을 확인한 결과, 5’arm 길이가 가장 짧고 CMV enhancer가 삽입 된 CMV-pPBC p-3378 벡터에서 활성이 가장 높게 나타났다. 이 벡터를 이용하여 hGH 유전자와 mRNA를 안정화시켜 유전자의 발현을 증가시킨다고 알려진 woodchuch hepatitis virus post-transcriptional regulator element (WPRE)를 함께 삽입하여 CMV-pPBC p-3378-hGH-WPRE 벡터를 구축하였다. 이 벡터로 transgenic mice를 생산하여 유선을 포함한 여러 조직으로부터 hGH의 발현을 RT-PCR 방법을 확인하였다. 그 결과 liver와 lung에서는 아주 약하게 hGH가 발현을 하였으나 유선 조직에서 hGH가 가장 많이 발현 하는 것을 볼 수 있었다. 형질전환 마우스 유즙 내의 hGH 발현을 western과 ELISA를 이 용하여 확인한 결과, 유즙 내에는 약 22 kDa의 hGH가 존재 하였으며 ml 당 최고 50 100 ug의 농도로 hGH를 포함하고 있었다. 이러한 결과들은 개량한 pPBC 벡터가 유선특 이적인 발현을 하며, 유선 세포주에서는 높은 활성을 나타냈지만 형질전환 마우스의 유 즙에서는 개량전의 벡터 보다 낮은 수준의 목적 단백질을 생산하여 서로 상이한 결과를 나타내었다. 따라서 앞으로도 유선특이적 발현 벡터인 pPBC 벡터의 개량 연구를 계속 진행할 것이다.

Generally, non-aflatoxigenic fungi, such as Aspergillus oryzae, and Aspergillus niger are main microflora in Korean traditional fermented foods including Meju and soybean paste, but sometimes, Aspergillus flavus and Aspergillus parasiticus can be contaminated and accumulated aflatoxins during fermentation and storage. So the screening of aflatoxigenic strains in fermented traditional food is very important to improve the sanitary quality of those foods. In this work, we screened aflatoxin producing fungi from commercial Meju and soybean paste in Western Gyeongnam by immunoassay. Samples were randomly purchased from market of the commercial Meju(10 EA) and soybean paste(20 EA) in nine areas of Western Gyeongnam. Of the samples collected, 24 strains and 22 strains of Aspergillus sp. were isolated from Meju and soybean paste, respectively. The isolated strains were cultured on SLS media at 25℃ for 15 days. The cultured broth were extracted with ethyl acetate and were analysed to determine aflatoxin B₁(AFB₁) by direct competitive ELISA(DC-ELISA). Six strains(25%) isolated from Meju, and 2 strains(9%) isolated from saybean paste, were confirmed as aflatoxin producing strains. The average range of aflatoxin productivity of isolates from Meju was 54.6 ± 38.7 ng/ml and that from soybean paste was 11.1 ± 8.6 ng/ml, respectively. Among them, isolated strain No. M-5-4 produced a high level of AFB₁ and showed 98.26 ng/ml of AFB₁. Every isolates were also re-confirmed their AFB₁ productivity by thin layer chromatography(TLC). The TLC results also showed same trend as DC-ELISA results. As the above results, the screening of hazard mycotoxigenic fungi from traditional fermented foods should be necessary for the safety and the application of HACCP system in the food manufactory in Korea.

This experiment was conducted to investigate the effects of mixed culture with mycotoxigenic and non-mycotoxigenic fungi on mycotoxin production. For this work, Aspergillus flavus (aflatoxin producing strain), Aspergillus niger (non-mycotoxigenic strain) and Penicilhum griseofulvum (patulin producing strainvere cultured in 5 ml SLS medium for 15 days under single or mixed culture. Affatoxin was determined by direct competitive ELISA, whereas, patulin was measured by HPLC. The mycelial growth, pH and total acidity were also observed by general methods. The mycelial growth was slightly decreased in the mixed culture, meanwhile total acidity was increased and pH was shown lower than that in single culture. Aspergillus flavus produced 145 ㎍/ml of aflatoxin for 12 days single culture, but in mixed culture, aflatoxin was decreased to 93%, and was shown as 10.16 ㎍/ml level. Patulin production in mixed culture was also decreased to 69.3% and was shown only 23.72 ㎍/ml level as compared with in single culture.

This study was designed to observe the effects of T-2 toxin on total cholesterol and lipid concentration in rat serum. T-2 toxin is a secondary metabolite produced by Fusa~rdum sp. which is often found on agricultural products including cereals, and it is a causal material of liver injuries in cattle and humans. When we fed rats with standard diet treated with T-2 toxin, the body weight and feed consumption of rats treated with T-2 toxin were decreased. As the results of lipid analysis, the concentrations of total cholesterol and free cholesterol in serum of treated rats were increased compared to non-fed control group, On the other hand, the levels of triglyceride and phospholipid in the serum of T-2 toxin treated experimental groups were declined. In conclusion, T-2 toxin largely influenced on the total cholesterol and lipid levels in rat serum.

Diethylnitrosamine (DEN) is known as a potential hepatic carcinogen by single administration. This study was designed to measure the effects of DEN-induced cell damage on the triglyceride and cholesterol concentration in the liver, excluding dietary effects. Fertilized chicken eggs, 10 days before hatching, were randomly divided into three groups (n=20) and each egg was injected 10 ul of corn oil (vehicle control), 5 ug of DEN/10 ul and 10 ug of DEN/10 ul into yolk via air sac. After 48 hr and 96 hr incubation, the damage of the chick-embryo liver cell was investigated by electron microscopy and by measuring the concentration of lipid components (total cholesterol, free cholesterol, phospholipid and triglyceride). For eggs administered 10 ug of DEN and incuvated 96 hr, in hepatocyte, the nucleus membrane was roughed, the size of nucleolus was apparently increased and euchromatin was accumulated. Mitochondria were condensed and cristae, located mitochondrial inner membrane, were obscured. Additionally, the levels of triglyceride and cholesterol classes were significantly increased depend on the amount of DEN and incubation time. Especially, triglycerides were notably increased in the group treated with 10 ug DEN at 96 hr, but phospholipids, component of cell membrane, were decreased with significance. As a conclusion, carcinogen induced hepatic lesion was correlated with the changes in lipid component of liver.

An enzyme-linked immunosorbent assay (ELISA) was established for the detection of zearalenone by using monoclonal antibodies produced by Z-M-26 hybridoma cells when injected into a mouse and zearalenone-oxime-OVA conjugate. Zearalenone-oxime-OVA conjugates were diluted with carbonyl buffer, coated to 96 well microtiter plates at 4℃ overnight and blocked with 1% BSA overnight. One thousand times diluted antibody solution together with standard zearalenone or sample was added to 96-well microtiter plates and stood overnight. A secondary antibody conjugated with HRP was added and an hour later, enzyme substrate (TMBZ) solution was added for color develpment. After 30 minutes, coloring reaction was terminated by adding 2 N H₂SO₄ and the O.D. was measured at 450 nm. Detection range of this method was about 0.1-100 ppb. The established indirect competitive ELISA method was suitable for a rapid and effective analysis of zearalenone in agricultural products.

The inhibitory effects of vegetables, collected from Jinju district, on the growth of Listeria monocytogenes were investigated. Among 12 vegetables, garlic, leek, onion and cabbage were remarkably effective to inhibit the growth of the strain. Especially with the addition of extract equivalent 0.25 g of raw garlic per ml broth, the population of the strain was decreased significantly, and completely inhibited with the addition of 0.625 g or more. During the incubation with the addition of extract equivalent 0.25 g of raw garlic per ml broth, the degree of inhibition was gradually increased with the lapse of time, and completely inhibited the growth of the strain after 48 hrs. While the pH value of treated broth remained unchanged almost, those of control were slightly reduced.