• 한국잠사곤충학회지
• /
• 제51권2호
• /
• pp.153-158
• /
• 2013

본 연구의 목적은 누에형질전환 기술을 이용하여 녹색형광실크를 개발하는 것으로서, 본 실험에서는 피브로인 H-chain의 N-말단과 C-말단을 이용하여 피브로인 재조합 단백질 발현 시스템을 제작하였고, 종결코돈이 없는 EGFP 유전자를 위의 발현 시스템에 클로닝하여 녹색형광실크를 제작하였다. 누에형질전환체 선발을 위해서는 3xP3 promoter와 DsRed2를 이용하여 선발하였고, 1200 개의 누에알에 microinjection 하여 F1 세대에서 8 bloods의 형질전환체를 선발하였다. 선발된 누에형질전환체는 초기배 단계의 눈과 신경조직, 유충과 번데기 그리고 성충의 눈에서 DsRed2 형광단백질이 발현되는 것을 확인 할 수 있었다. 또한 실크의 피브로인에서 EGFP 단백질이 발현되는 것을 확인하기 위해, F2세대의 누에형질전환체 중에서 5령 3일 유충을 해부하여 견사선을 형광현미경으로 관찰하였고, 중부와 후부 견사선에서 형광단백질이 발현되는 것을 확인할 수 있었다. 그리고 F2 세대의 고치와 저온에서 정련한 실크에서도 녹색형광단백질의 발현을 확인할 수 있었고, Western blot 분석에서도 EGFP 재조합 단백질이 피브로인 H-chain과 융합된 형태로 존재하는 것이 확인되었다. 이상의 결과에서 녹색형광실크를 생산하는 누에형질전환체가 성공적으로 제작 되었음을 확인할 수 있었고 이러한 결과를 토대로 새로운 산업소재로서 실크를 활용할 수 있을 것으로 기대한다.

• 한국잠사곤충학회지
• /
• 제49권2호
• /
• pp.67-70
• /
• 2007

실크단백질 식이공급이 동물대장암에 미치는 효과를 알아본 결과 다음과 같은 결과를 얻을 수 있었다. 1. 동물 대장암에 투여한 식이공급용 선발 실크단백질의 피브로인 및 세리신 단백질은 각각 Mw 65,000 내외의 피브로인 및 Mw 100,000만 내외의 세리신 단백질이며, 동물사료의 경우, 섭식 등 물성 등이 작년대비 개선된 3% 첨가된 전용 사료를 조제하여 섭식하도록 하였다. 2. 1,2-DMH에 의한 동물 대장암 유발(1회/1주, 4회 주사)후 6주 시료 투여에 있어서 대장암 유발군(CA)의 경우, 체중변화 및 식이 섭취량의 관계인 식이효율은 세리신 섭취군(S)의 경우 정상군(C)의 85% 수준을 나타내었다. 3. 동물대장암 조직의 조직관찰에 있어서는 대장암 조직의 COX-2 발현을 염색한 결과, 대장암 유도군(CA)의 경우 확실한 COX-2에 의한 발현을 나타내었으며, 세리신 섭취군(S)의 경우는 그 정도가 미비함을 나타내어 대장암에 효과가 있음을 나타내었다. 4. 대장암 인자변화를 정량적으로 확인한 결과, 정상군(C)에 비하여 대장암 유발군(CA)은 확실한 7배 정도의 COX-2량이 확인되었으며, 세리신 섭취군(S)의 경우는 유발군(CA) 대비 1/2 이하 수준으로 줄어듬을 확인할 수 있어서 금후 대장 질환 관련 소재 개발 가능성을 나타내었다.

• 생약학회지
• /
• 제33권1호통권128호
• /
• pp.21-28
• /
• 2002

Antidiabetic effects of the acid hydrolysate of silk fibroin were investigated by oral administration to animal models for diabetes mellitus, Fibroin protein was extracted from cocoon and digested to peptides of low-molecular weight range (mainly below 3,000) and amino acids by acid hydrolysis, Feeding of the fibroin hydrolysate resulted in a significant recovering effect on reduction of body weight gain and a lowering effect on blood glucose gain in streptozotocin-induced diabetic Sprague Dawley rats (STZ rats) which were used as an insulin-dependent diabetic animal model. But the body weight and blood glucose level in C57BL/KsJ-db/db mice (db/db mice), an non-insulin-dependent diabetic animal model, were not changed significantly by the feeding, On the other hand, plasma leptin levels increased according to increased feeding amount of the hydrolysate in STZ rats and db/db mice in common, It was concluded from the results that the fibroin hydrolysate might stimulate the insulin secretion by recovering or activating pancreatic ${\beta}$ cells and result in the increased plasma leptin level. It was also deduced that the antidiabetic improvements in body weight and blood glucose gain in STZ were thought to be due to the increased insulin secretion, but in db/db mice of which the diabetic symptoms were caused by insulin resistance, the stimulated secretion of insulin was unlikely to be able to change body weight and blood glucose level significantly.

• 한국잠사곤충학회지
• /
• 제38권2호
• /
• pp.160-167
• /
• 1996

견단백질을 구성하는 액상견사막인 중부견사막을 추출한 후, 약3배로 급속히 연신하여 견 monofilament를 제작하였다. 이 시료를 연화시켜 분자간의 수소결합능을 저하시키기 위해 비등상태의 증류수에 팽윤시킨 다음 연신처리를 행한 결과는 다음과 같다. 1. 열분석과 X선회석의 결과로부터 견사막건조물시료은 Silk I형의 결정영역 및 random 무정형분자로 구성되어 있었으나 monofilament시료에서는 부분적으로 Silk II형결정이 발현하기 시작하여 더욱더 연신한 62.5% 연신시료에서는 거의 완전한 Silk II형결정으로 전이하는 것을 알 수 있다. 2. 복굴절율 및 밀도는 monofilament시료에 비해 연신율의 상승과 함께 증가하는 경향을 나타내어 견 fibroin분자의 배향은 초기에 급격히 증가하여 fibroin분자의 배향이 완성되었다. 3. 가수분해처리에 의한 복굴절율은 연신율의 상승에 따라서 증가하는 경향이었다. 또, 가수분해의 처리시간과의 관계에서는 연신율이 증가함에 따라 복굴절율도 거의 일정하게 나타나 배향결정화가 진행되어 배열이 좋게 되어진 무정형이 결정으로 전이하는 것이 시사되었다. 4. 62.5% 연신시료를 가수분해한 경우의 X선회절은 가수분해에 의해 배향의 흐트러짐은 생기나 결정풍괴는 일어나지 않았다. 열분석에서도 가수분해의 처리시간이 증가함에 따라 최대흡열 peak 온도는 저온측으로 이동하여 가수분해처리에 의해 고배향의 무정형부분이 유출로 인하여 결정부분의 배향이 흐트러진다는 것이 X선분석 결과로부터 추측되어진다.

• 한국섬유공학회:학술대회논문집
• /
• 한국섬유공학회 2003년도 The Korea-Japan Joint Symposium
• /
• pp.88-89
• /
• 2003

A series of novel spinning acrylic polymer containing silk protein were synthesized by copolymerization of acrylonitrile (AN) and silk fibroin peptide (SFP) modified by acryloyl chloride (AC) with vinyl groups. The viscosity values of these copolymers showed that the copolymers have good spinnability, which are synthesized under the condition of putting a micro amount of metal ions into synthesizing solvent. The fiber based on the poly (AN-co-SFP) was prepared and characterized by SEM, FTIR measurement of its shell and core flakes, and moisture absorption. The fiber appeared a smooth surface and could be assumed to have excellent adhesive between SFP and PAN. Furthermore, these fibers showed a shell-core structure and excellent moisture absorption.

• 한국잠사곤충학회지
• /
• 제53권2호
• /
• pp.92-96
• /
• 2015

본 연구는 장려 품종 누에고치로부터 실크 세리신과 실크 피브로인으로 분리하여 이들 단백질을 세포 첨가제로 개발하기 위하여 수행되었다. 장려품종 누에고치를 고온 고압기를 통하여 실크 세리신을 먼저 추출하였으며, 남겨진 실크 피브로인을 이용하여 시간별로 용해하여 품종별 실크 피브로인을 얻었다. 이들 실크 세리신과 실크 피브로인을 이용하여 세포 독성, 세포 증식 및 분열 관련 유전자 발현 분석을 확인하였다. 먼저, 누에고치의 특성을 분석하기 위하여 공극률을 측정하였다. 공극률은 금옥잠 누에고치의 경우 84.71%, 대성잠 81.58%, 백옥잠 73.23%의 공극률 값이 나타났다. 분자량 차이에 있어서 실크 세리신은 품종의 영향이 크지 않았으나 실크 피브로인의 경우, 금옥잠은 5시간의 장시간의 용해에도 불구하고 100 kDa 이상의 큰 분자량을 나타내었다. 장려품종 누에를 사용하여 제조된 실크 세리신과 실크 피브로인을 이용하여 세포 증식 실험을 하였으며 백옥잠으로부터 얻은 실크 세리신의 경우 농도에 따라 유의적으로 세포 증식 효과가 나타났으며, 금옥잠으로부터 얻은 실크 피브로인의 경우 5시간 용해 시 세포 증식에 있어서 가장 우수한 결과를 보였다. 또한, 백옥잠 실크 세리신 처리 결과 세포 증식 관련 유전자의 발현수준이 증가됨을 확인할 수 있었다.

• Journal of Nutrition and Health
• /
• 제45권2호
• /
• pp.113-120
• /
• 2012

In our previous studies, dietary supplements of silk protein, sericin, and fibroin, were beneficial for improving epidermal levels of ceramides, which are the major lipids for maintaining the epidermal barrier. In this study, we investigated the dietary effects of silk protein on epidermal levels of free sphingoid bases and their phosphates such as $C_{18}$ sphingosine (So), $C_{18}$ sphinganine (Sa), $C_{18}$ sphingosine-1-phosphate (S1P), and $C_{18}$ sphinganine-1-phosphate (Sa1P), which are either synthetic substrate or degradative metabolites of ceramides. Forty-five male NC/Nga mice, an animal model of atopic dermatitis (AD), were divided into three groups: group CA was an atopic control and fed a control diet, group S was fed a 1% sericin diet, and group F was fed a 1% fibroin diet. Fifteen male BALB/c mice served as group C (control group) and were fed the control diet. All mice were fed with diets and water $ad$ $libitum$ for 10 weeks. Sa in group CA was lower than that in group C, but So in group CA was similar to that in group C. So and Sa were higher in groups S and F than those in group CA; So level was even higher than that in group C, and Sa level was similar to that of group C. The So/Sa ratio in group CA, which is reported to increase in AD, was significantly higher than that of group C. The So/Sa ratio was lower in groups S and F than that in group CA, and decreased further in group F. However, S1P and Sa1P in groups S and F were similar to those in group CA. Taken together, we demonstrated that silk protein, sericin and fibroin dietary supplements, increased So and Sa levels, and decreased the So/Sa ratio.

• International Journal of Industrial Entomology and Biomaterials
• /
• 제5권1호
• /
• pp.93-102
• /
• 2002

We have cloned and characterized the complete fibroin light chain gene from the silkworm Baekok-Jam, Bombyx mori, a recommended variety in Korea. It consists of seven exons and six introns. It consists of 14,663 nucleotides long with an open reading frame of 786 nucleotides that encodes a protein of 262 amino acid residues with a molecular mass of approximately 26,000 dalton. The amino acid alignment revealed that the Baekok-Jam fibroin light chain had 98.5 % protein sequence identity to J139 strain: differed at four amino acid positions (11, 46, 80 and 123). The Northern hybridization analysis showed that the Baekok-Jam fibroin light chain gene was specifically expressed in the posterior silk gland.

• International Journal of Industrial Entomology and Biomaterials
• /
• 제17권2호
• /
• pp.229-234
• /
• 2008

We used serial analysis of gene expression (SAGE) approach to derive a profile of expressed genes of the posterior silk glands (PSG) and to create a reference for understanding gene cluster related to the mechanism of silk protein synthesis in the silkworm, Bombyx mori. We constructed a 3' SAGE library from the PSG of the fifth instar larvae of the silkworm. In total we obtained 2,406 SAGE tags, of which 682 were unique tags. Sorted by tag count number, 27 (4%) unique tags were significantly more abundant genes (ten or more times), whereas 445 (65%) unique tags were detected as single copies. The annotation of 682 unique SAGE tags revealed that 462 (68%) of the SAGE tag sequences represented known genes, whereas 220 (32%) of the tag sequences had no matches in SAGE map and silkworm EST databases. Of the 682 SAGE tags, the most abundant tag sequences were that of the fibroin light chain gene and the silk protein P25. In addition, we compared two relative abundance results of the SAGE and the EST approaches to verify whether their transcript quantitative aspects are significant or not. The comparative results of relative abundances of the fibroin H-, L- chain and P25 glycoprotein genes indicated that the quantitative approach based on SAGE tags is effective for quantitative cataloging and comparison of expressed genes in same organs. The SAGE tag information reported in this study would be useful for researchers in the field to analyze genes associated with silk processing mechanisms of insects.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
• /
• 제36권5호
• /
• pp.366-374
• /
• 2010

Introduction: This study evaluated the capability of silk fibroin (SF) and recombinant human bone morphogenetic protein-2 loaded SF (SF-BMP) as a bone defect replacement matrix when grafted in a calvarial bone defect of rats in vivo. Materials and Methods: A total 70 calvarial critical size defects (5.0 mm in diameter) made on 35 adult female Sprague-Dawley rats were used in this study. The defects were transplanted with (1) rhBMP-2 loaded silk fibroin graft (SF-BMP: 0.8+$10\;{\mu}g$), (2) Silk fibroin (SF: $10\;{\mu}g$), and (3) no graft material (Raw). The samples were evaluated with soft x-rays, alkaline phosphatase activity, calcium/phosphate quantification, histological and histomorphometric analysis at postoperative 4 and 8 weeks. Results: The SF-BMP group ($48.86{\pm}14.92%$) had a significantly higher mean percentage bone area than the SF group ($24.96{\pm}11.01%$) at postoperative 4 weeks.(P<0.05) In addition, the SF-BMP group ($40.01{\pm}12.43%$) had a higher % bone area at postoperative 8 weeks than the SF group ($33.26{\pm}5.15%$). The mean ratio of gray scale levels to the host bone showed that the SF-BMP group ($0.67{\pm}0.08$) had a higher mean ratio level than the SF group ($0.61{\pm}0.09$) at postoperative 8 weeks. These differences were not statistically significant.(P=0.168 and P=0.243, respectively) The ratio of the calcium and phosphate contents of the SF-BMP ($0.93{\pm}0.22$) group was lower than that of the SF ($1.90{\pm}1.42$) group at postoperative 4 weeks. However, the SF-BMP group ($0.75{\pm}0.31$) had a higher Ca/$PO_4$ ratio than the SF ($0.68{\pm}0.04$) at postoperative 8 weeks. These differences were not statistically significant.(P=0.126 and P=0.627, respectively) For the bone-specific alkaline phosphatase (ALP) activity, which is recognized as a reliable indicator of the osteoblast function, the SF-BMP ($23.71{\pm}8.60\;U/L$) groups had a significantly higher value than the SF group ($12.65{\pm}6.47\;U/L$) at postoperative 4 weeks.(P<0.05) At postoperative 8 weeks, the SF-BMP ($21.65{\pm}10.02\;U/L$) group had a lower bone-specific ALP activity than the SF group ($16.72{\pm}7.35\;U/L$). This difference was not statistically significant.(P=0.263) For the histological evaluation, the SF-BMP group revealed less inflammation, lower foreign body reactions and higher bone healing than the SF group at postoperative 4 and 8 weeks. The SF group revealed more foreign body reactions at postoperative 4 weeks. However, this immunogenic reaction decreased and the remnant of grafted material was observed at postoperative 8 weeks. For histomorphometric analysis, the SF-BMP group had a significantly longer bone length to total length ratio than those of the SF group at postoperative 4 and 8 weeks.(P<0.05) Conclusion: The rhBMP-2 loaded silk fibroin graft revealed fewer immunoreactions and inflammation as well as more new bone formation than the pure silk fibroin graft. Therefore, silk fibroin may be a candidate scaffold for tissue engineered bone regeneration.