시스타틴(cystatin: CST)은 C1A류 시스테인 단백질분해효소에 대한 경쟁적 가역억제자로서 동식물류에서 파파인과 같은 캐셉신을 억제 대상으로 작용하게 된다. 바이러스 유래 CST (CpBV-CST1)이 폴리드나바이러스의 일종인 CpBV (Cotesia plutellae bracovirus)에서 동정되었 다. 기존 연구는 이 유전자의 과발현이 배추좀나방(Plutella xylostella) 유충의 면역 및 발육을 교란한다는 것을 보여 주었다. 본 연구는 이 유전자 의 단백질 기능을 분석하기 위해 세균발현시스템을 이용하여 재조합단백질(rCpBV-CST1)을 형성하여 단백질분해효소에 대한 활성억제효과를 결정하고, 곤충의 면역과 발육에 대한 생리적 억제효과를 분석했다. 이 유전자 번역부위는 138 개 아미노산으로 약 15 kDa 크기의 단백질로 추 정되었다. CpBV-CST1이 먼저 pGEX 발현벡터에 재조합되고, BL21 STAR (DE3) competent cells에 형질전환된 후 0.5 mM IPTG로 4 시 간동안 과발현되었다. 분리된 재조합단백질은 파파인에 대한 뚜렷한 억제효과를 나타냈다. 이 재조합단백질은 파밤나방(Spodoptera exigua)에 대 해서 혈구소낭형성의 세포성 면역반응을 억제하고, 경구로 처리할 때 배추좀나방의 유충발육을 처리 농도에 비례하여 제한시켰다. 이상의 결과 는 CpBV-CST1이 해충 밀도 억제에 응용될 수 있음을 제시하고 있다.

돼지 써코바이러스 2형(porcine circovirus type 2)은 단일가닥 원형DNA바이러 스이며 돼지에 심각한 질병을 일으키는 돼지이유자돈전신성소모성 증후군의 주요 한 인자로 알려져 있다. 돼지 써코바이러스 2형은 2개의 주요한 ORF를 가지고 있 으며 ORF1은 바이러스의 복제, ORF2는 캡시드단백질의 형성에 관여한다. 이 중, ORF2의 해독에 의해 생성된 캡시드단백질은 바이러스의 구조형성 뿐만 아니라 항 원단백질로써 알려져 있으며, 이에 따라 본 연구에서는 베큘러바이러스 다각체 단 백질과 부분 융합에 의한 돼지 써코바이러스 2형 구조단백질의 재조합 발현을 확 인하였다. 발현된 단백질은 SDS-PAGE 상에서 분석하였고, 항-돼지 혈청, 항 -PCV2 ORF2 단일항체 그리고 히스텍 항체를 사용하여 Western blot 분석으로 확 인하였다. 그 결과 재조합 단백질은 재조합 바이러스 접종 후 2일째부터 발현이 나 타나며 4일째 최대 발현하는 것을 확인하였다. 또한 다각체 단백질과 부분 융합한 바이러스는 비융합 바이러스보다 재조합 단백질 생산이 증대 되었으며, 이는 다각 체 단백질과 부분 융합을 이용한 돼지 써코바이러스 2형 구조단백질 ORF2의 발현 을 향상 시킬 수 있음을 보여준다.

Abnormal prions are infectious agents involved in a neuro-degenerative disease, which occurs naturally such as Chronic Wasting Disease (CWD) in deer and elk, Bovine Spongiform Encephalopathy (BSE) in cattle, Scrapie in sheep and goats and Creutzfeldt-Jakob Disease (CJD) in humans. The cellular prion protein of the elk consists of 233 amino acids (residues 25-257), which represents an autonomous folding unit with three α-helices and two-stranded anti-parallel β-sheets. Here, we demonstrated elk-recPrP (Elk recombinant prion protein) which can be obtained as follows; (1) Cloning of elk PrP gene, (2) Expression of a histidine-tagged full-length elk PrP by induction with IPTG in E. coli and (3) Purification by affinity chromatography using Ni-NTA agarose resin. In Western blot and ELISA analysis, elk-recPrP showed specific activity against anti-PrP monoclonal antibody. Thus, our elk-recPrP would be a useful tool for the understanding of basic structure and mechanism studies of PrPSC formation.

The regeneration of periodontium is the goal of periodontal therapy. Many periodontologists try to achieve this goal by using guided tissue regeneration(GTR) method. However, these procedures always include several disadvantages. Recombinant human bone morphogenetic protein-2 (rhBMP-2) stimulated ectopic bone formation when it was implanted in rat muscles with insoluble bone matrix by differentiating muscle cells into chondrocytes and osteoblasts. The purpose of this study was to evaluate the osteoinductive potential of the mixture of rhBMP-2 (5 μg/ml) and heparin (0.25 or 25 μg/ml ) at the critically sized rabbit calvarial defects. And this study aimed to reveal that heparin also acts to enhance the bone forming activity of rhBMP-2. The 12 rabbits (4-month-old; NewZealand White) were used in the present study. 5 μg/ml of rhBMP-2 and 0, 0.25 or 25 μg/ml of heparin were mixed and blotted into anorganic bovine bone and filled cranial defects. The animals were sacrificed following a time schedule (1, 3, and 6 weeks). Sections were made in 7 μm thicknesses, stained with H&E and Masson's trichrome method, and examined under a light microscope. The differences among each obtained percent value were evaluated by one-way analysis of variance. A p value of p<0.05 was considered statistically significant and an ANOVA test was performed to verify significant differences. To adjust for multiple comparisons when one-way analysis of variance showed a significant difference between groups (p<0.05), Scheffe`s post hoc test was used to identify which group differences accounted for the significant p-value. In control group, osteoinduction was not outstanding, however, in experimental groups, osteoinduction was significantly outstanding, and as the concentration of heparin mixed with rhBMP-2 increased, osteoinduction was increased. Mixtures of rhBMP-2 and heparin affect bone formation at initial bone formation, but that effect disappeared following a time lapse.

Bone morphogenetic proteins (BMPs) are known to promote osteogenesis, and clinical trials are currently underway evaluating the ability of BMPs to promote bone formation in grafting procedures and fracture healing. Some studies, have independently reported that sulfated polysaccharides particularly heparin, enhance the osteoblastic differentiation induced by BMPs in vitro, and another study demonstrated that heparin enhanced the bone formation induced by BMP‐2 in vivo. This study was performed to examine adipose stem cell responses to rhBMP‐2 alone and rhBMP‐2 with heparin at 0.25, and 25 μg/㎖ concentrations, respectively, in culture media. Adipose stem cells were cultured for 2, 4, and 8 days toward the osteoblastic differentiation in rhBMP‐2 alone and rhBMP‐2 with heparin at 0.25, and 25 μg/㎖ concentrations, respectively, in culture media. Verification of the stem cell lineage was performed in two ways. The first method was a continuous sequential culture until 5th generation. The second method was using monoclonal antibodies for STRO‐1 and CD 90. Naphthol AS phosphate‐fast blue BB staining for alkaline phosphatase was used for verifying osteoblastic differentiation because Alkaline phosphatase activity had been used as an osteoblastic differentiation marker and degree of osteoblastic activity. Alizarin red staining was also used as an osteoblastic differentiation marker because it quantifies the calcium levels in cells or tissues. During the 5th generation culture, cultured cells actively proliferated, and these cultured cells showed a positive reaction to STRO‐1 and CD90 cell surface molecules. Naphthol AS phosphate‐fast blue BB staining and Alizarin red staining were positive in most samples of each group at 2, and 4 days and positive reaction was proportioned to degree of morphological differentiation. In the concentration of 25 μg/ml of heparin, the ALP activity was highest at the 2nd day in the culture, and then the activities of ALP were decreased significantly at 4, and 8 days. The ALP activity was greatest at the 4th day of the culture, and then decreased significantly at the 8th day in 0 μ g/ml and 0.25 μg/ml of heparin concentrations, Adipose stem cells could be differentiated in rhBMP‐2 in culture media, and the addition of heparin to BMP‐2 promoted differentiation of osteoblasts. Moreover, morphological differentiation was associated with the activity of osteoblasts. This study was shown that, when heparin concentration increases, the early differentiation of the cells was brought about, but the early differentiated cells were rapidly progressed to degenerative changes

단백질분해효소를 생산하지 않는 균주 B. subtilis MT-2의 염색체 DNA를 추출한 다음, B. subtilis AC819 균주에 상동성 유전자재조합을 이용하여 competent cell 형질전환을 시켰다. 얻어진 형질전환체를 B. subtilis HL-1이라고 명명하였으며, 그 표현형은 histidine 요구성, streptomycin 내성, tetracyclin 내성을 나타내면서 단백질 분해효소를 생산하지 않았다. 플라스미드 pUB11