경북대학교병원 치과 내원환자들의 특성 및 만족도 변화추이

정성화 ( Seong Hwa Jeong ),김지영 ( Ji Young Kim ),이원기 ( Won Kee Lee ),손은영 ( Eun Young Son ),최연희 ( Youn Hee Choi ),송근배 ( Keun Bae Song ) 대한구강보건학회 2004 大韓口腔保健學會誌 Vol.28 No.2

실크단백질 효소 가수분해물이 OLETF Rat의 혈당, 혈중 인슐린과 렙틴분비에 미치는 영향

이영숙,박민정,최지은,김지영,남문석,정윤화,Lee, Young-Sook,Park, Min-Jeong,Choi, Ji-Eun,Kim, Ji-Young,Nam, Moon-Suk,Jeong, Yoon-Hwa 한국식품영양과학회 2007 한국식품영양과학회지 Vol.36 No.6

본 연구는 누에고치를 가수분해하여 얻은 실크단백질 효소 가수분해물이 비만형 당뇨병 모델인 OLETF 쥐의 당뇨병 개선에 미치는 영향을 조사하였다. 27주령의 OLETF 쥐를 당뇨대조군과 실크단백질 효소 가수분해물 0.5%, 0.8% 섭취군으로 나누어 19주 동안 음수로 섭취시켰다. 19주 동안 실험동물의 체중, 식이 섭취량, 음수 섭취량을 측정하고, 매주 2회씩 비공복과 공복 혈당변화를 관찰하였으며, 19주 후 모든 동물을 희생시킨 후 혈액을 채취하여 혈청지질과 인슐린 및 렙틴의 농도를 분석하였다. 당뇨대조군의 체중은 실크단백질 효소 가수분해물 섭취군에 비해 크게 감소하는 경향을 보였고, 총 콜레스테롤은 농도 의존적으로 그 수치가 낮아지는 경향을 보였으나 유의적 차이는 없었다. 또한 중성지질이나 HDL-cholesterol 함량 변화에도 큰 영향을 미치지 못하였다. 실크단백질 효소 가수분해물 섭취군은 대조군에 비하여 혈당 상승이 유의적으로 억제되었다. 17주 후 내당능 측정결과 실크단백질 효소 가수분해물 섭취군의 최고 혈당치가 농도 의존적으로 낮게 나타나는 경향을 보였으며 회복도 빨랐다. 인슐린과 렙틴은 농도 의존적으로 증가하였으며, 유의적인 차이를 보였다. 실크단백질 효소 가수분해물의 섭취는 인슐린과 렙틴의 대사에 관여하여 혈당상승을 억제하는 것으로 사료된다. This study was performed to investigate the effect of silk protein hydrolysates hydrolyzed by protease on blood glucose level, serum insulin and leptin secretion in the OLETF rats. Twenty seven week-old-male OLETF rats were divided in three groups: diabetic control, 0.5% and 0.8% silk protein hydrolysates groups that were fed daily for 19 weeks. Body weight increased in the 0.5% and 0.8% silk protein hydrolysates fed groups compared with diabetic control group. Food and water intake were not different among diabetic and silk protein hydrolysates groups. In random state, the blood glucose levels in silk protein hydrolysates fed groups were lower than diabetic control group; however, the blood glucose in the three groups were not different in fasting state. Also silk protein hydrolysates improved the glucose tolerance in OLETF rat. The silk protein hydrolysates did not influence serum lipids while serum insulin and leptin levels were increased in the experimental OLETF rats. These results suggested that the administration of silk protein hydrolysates solution reduced significantly (p<0.05) an increasing rate of blood glucose level by stimulating the insulin secretion and increasing the serum leptin level.

사람 프로트롬빈을 코딩하는 전체 cDNA 의 클로닝 및 원숭이 COS 세포에서의 발현

김종명,김용주,최은화,김재만,김지영 ( Jong Myoung Kim,Yong Joo Kim,Eun Hwa Choi,Jae Man Kim,Ji Young Kim ) 생화학분자생물학회 1990 BMB Reports Vol.23 No.4

cDNA fragments coding for the leader sequence and N-terminal region of human prothrombin was isolated from partial cDNA clones that were enriched for prothrombin cDNA clones. Poly (A)+ RNA was isolated from human liver and size-fractionated by sucrose gradient centrifugation. Poly (A) + RNAs in the ranges of 1500 to 4000 nucleotides were pooled and utilized for cDNA synthesis by reverse transcription using a specific oligodeoxynucleotide as a primer. Among about 2000 colonies, eight positve cDNA clones were obtained by hybridization with ^(32)P-labeled oligodeoxynucleotide complementary to 5`-terminal region of prothrombin mRNA sequences. Six of the recombinant plasmids contained prothrombin cDNA inserts of expected size. Restriction and sequencing analysis showed that the cDNA insert of phII3 plasmid contained 12 nucleotides of 5`-untranslated region and the DNA sequences conding for the leader and N-terminal 312 residues of human prothrombin. The complete cDNA sequence coding for the leader and 579 amino acid residues of human prothrombin was cloned in pUC18 by combining the two overlapping cDNA inserts of phII3 and pHPT2 (Choi et al., 1989). The full-length prothrombin cDNA was inserted into the multicloning sites downstream from the SV40 late promoter of pSLS plasmid. Upon transfection with the recombinant plasmid, human prothrombin was expressed in SV40-transformed African green monkey kidney(COS) cells, and was exclusively found in the growth medium.

E . coli 에서 사람 프리트롬빈 2 cDNA 의 크로닝과 발현

최은화,김용주,김종명,홍효정,한문희,김지영 ( Eun Hwa Choi,Yong Joo Kim,Jong Myoung Kim,Hyo Jeong Hong,Moon Hi Han,Ji Young Kim ) 생화학분자생물학회 1989 BMB Reports Vol.22 No.2

A 0.6 kbp of 3`-portion of human prothrombin cDNA was isolated from a liver cDNA library of λgt 10 by in situ plaque hybridization. A cDNA fragment corresponding to the N-terminal region of prethrombin 2 was obtained from another cDNA library synthesized by reverse transcription of human liver mRNA using a specific oligonucleotide as a primer. The 48 bp cDNA sequences starting from the first nucleotide to the AvaI site of human prethrombin 2 cDNA was chemically synthesized such that prethrombin 2 can be expressed directly in E. coli expression vector system. The open reading frame(ORF) for human prethrombin 2 was cloned in pUC18 by combining the synthetic fragment and the cDNA fragments. Expression plasmids were constructed in which the ORF for human prethrombin 2 was inserted into the site downstream from the thermoinducible leftward promoter (P_L) and the cII ribosome binding site of bacteriophage λ. Upon temperature induction, the plasmids expressed 34,000 dalton polypeptide, which was accumulated to an amount corresponding to 5-10% of the total bacterial proteins. The 34,000 dalton polypeptide was identified as human prethrombin 2 by SDS-gel electrophoresis and Western blot analysis.

Cloning and expression of human prethrombin 2 cDNA in Escherichia coli

최은화,김용주,김종명,홍효정,한문희,김지영,Choi, Eun-Hwa,Kim, Yong-Joo,Kim, Jong-Myoung,Hong, Hyo-Jeong,Han, Moon-Hi,Kim, Ji-Young 생화학분자생물학회 1989 한국생화학회지 Vol.22 No.2

A 0.6 kbp of 3'-portion of human prothrombin cDNA was isolated from a liver cDNA library of ${\lambda}gt$ 10 by in situ plaque hybridization. A cDNA fragment corresponding to the N-terminal region of prethrombin 2 was obtained from another cDNA library synthesized by reverse transcription of human liver mRNA using a specific oligonucleotide as a primer. The 48 bp cDNA sequences starting from the first nucleotide to the AvaI site of human prethrombin 2 cDNA was chemically synthesized such that prethrombin 2 can be expressed directly in E. coli expression vector system. The open reading frame(ORF) for human prethrombin 2 was cloned in pUC18 by combining the synthetic fragment and the cDNA fragments. Expression plasmids were constructed in which the ORF for human prethrombin 2 was inserted into the site downstream from the thermoinducible leftward promoter $(P_L)$ and the cII ribosome binding site of bacteriophage ${\lambda}$. Upon temperature induction, the plasmids expressed 34,000 dalton polypeptide, which was accumulated to an amount corresponding to 5-10% of the total bacterial proteins. The 34,000 dalton polypeptide was identified as human prethrombin 2 by SDS-gel electrophoresis and Western blot analysis. 사람 간 cDNA library로부터 프로트롬빈 mRNA의 3'쪽에 해당하는 0.6kbp cDNA를 plaque hybridization에 의해 분리하였다. 프로트롬빈 2의 N-말단에 해당하는 cDNA 절편은 간 mRNA와 상보적인 올리고뉴클리오티드 primer를 이용하여 만든 다른 cDNA library로부터 분리하였다. 프로트롬빈 2를 E. coli에서 직접 발현시키기 위해 프리트롬빈 2cDNA의 첫 뉴클라오티드부터 Ava I 부위까지의 48bp DNA를 화학적으로 합성하였다. 합성 DNA와 cDNA 절편들을 이용하여 완전한 사람 프리트롬빈 2 번역프레임 (ORF)를 포함한 pUC18 을 클로닝하였다. 사람 트리트롬빈 2 ORF를 박테리오파지 ${\lambda}$의 $P_L$ promoter와 c II ribosome binding site 아래에 연결하여 프리트롬빈 2 발현벡터를 제조하였다. 온도에 의해 유도시킨 결과 이 플라스미드는 E. coli에서 약 34,000 dalton의 폴리펩티드를 생성하였다. 이 폴리펩티드는 전체 박테리아 단백질의 5-10% 정도로 만들어졌다. Western blot 분석에 의해 이 폴리펩티드가 사람 프리트롬빈 2 라는 것을 확인하였다.

Cloning of Full-length cDNA Coding for Human Prothrombin and Its Expression in Monkey COS Cells

Kim, Jong-Myoung,Kim, Yong Joo,Choi, Eun-Hwa,Kim, Jae-Man,kim, Jiyoung 경희대학교 유전공학연구소 1990 遺傳工學論文集 Vol.2 No.-

프로트롬민 cDNA 클론이 많이 함유된 partial cDNA clone들로부터 프로트롬빈 신호서열 및 N-말단 부위를 코딩하는 cDNA를 분리하였다. poly(A)+RNA는 사람 간 조직에서 분리하여 sucrose gradient centrifugation에 의해 분획하였다. 1,500에서 4,000 뉴클리오티드 사이의 mRNA를 모아 역전사에 의한 cDNA 합성에 이용하였다. 프라이머로는 프로트롬빈 mRNA에 상보서열을 가지는 올리고뉴클리오티드를 사용하였다. 약 2,000개의 콜로니 중에 8개가 N-말단쪽에 해당하는 올리고뉴클리오티드 프로브와 반응하였다. 이 중 여섯 개의 플라스미드가 예상한 길이의 프로트롬빈 cDNA insert를 함유하고 있었다. 제한효소 및 염기서열 분석 결과 phI13 플라스미드는 프로트롬빈 N-말단쪽 312개 아미노산과 43개의 신호서열을 코딩하는 부위 및 12 뉴클리오티드의 5'-untranslated sequence를 포함하고 있음을 보여주었다. 이 5'-쪽 cDNA와 pHPT2(Chot et al., 1989)의 cDNA를 연결하여 신호서열 및 579 아미노산을 코딩하는 전체 cDNA를 클로닝하였다. 프로트롬빈 cDNA를 SV 40 바이러스 후기 프로모터 아래에 연결하여 포유배양세포발현 플라스미드를 제조하였다. 이 플라스미드를 원숭이 COS 세포에 넣었을 때 프로트롬빈이 발현되었으며 발현된 프로트롬빈은 세포의 배양액에서 발견되었다. cDNA fragments coding for the leader sequence and N-terminal region of human prothrombin was isolated from partial cDNA clones that were enriched for prothrombin cDNA clones. Poly (A)+RNA was isolated from human liver and size-fractionated by sucrose gradient centifugation. Poly(A)+RNAs in the ranges of 1500 to 4000 nucleotides ere pooled and utilized for cDNA synthesis by reverse transcription using a specific o1igodeoxynucleotide as a primer. Among about 2000 colonies, eight positive cDNA clones are obtained by bridization with (32)^P-labeled oligodeoxynucleotide complementary to 5'-terminal region of prothrombin mRNA sequences. Six of the recombinant plasmids contained prothrombin cDNA inserts of expected size. Restriction and sequencing analysis showed that the cDNA insert of phII3 plasmid contained 12 nucleotides of 5'-untranslated region and the DNA sequences conding for the leader and N-terminal 312 residues of human prothrombin. The complete cDNA sequence coding for the leader and 379 amino acid residues of human prothrombin was cloned in pLC18 by combining the two overlapping cDNA inserts of phII3 and pHPT2 (Chor et al., 1989) The full-length prothromibin cDNA was inserted into the multicloning sites don nstream from the SV40 late promoter of pSLS plasmid. Upon transfection with the recombinant plasmid, human prothrombin was expressed in SV40-transformed African green monkey kidney(COS) cells, and was exclusively found in the growth medium.

Alteration of the Nucleotide Sequence Preceding the Translation Initiation Codon and the Effects on the Expression of Human Prethrombin 2 in E. coli

Kim, Yong-Joo,Lee, Seung-Cheol,Kim, Jae-Man,Kim, Jong-Myoung,Choi, Eun-Hwa,Kim, Ji-Young 경희대학교 유전공학연구소 1989 遺傳工學論文集 Vol.1 No.-

전에 발표된 논문에서 사람 프리트룸빈 2를 높은 수준으로 생산하는 재조합 플라스미스 pASPT2 제소에 관해 보고한 바 있다(Chot et al., 1989). pASPT2에서 사람 프리트롬빈 2 유전자는 박테리오파지 λP_(L) 프로모터에 의해 발현이 조절되는데 세포내에서 전체 박테리아 단백질의 10% 정도까지 그 유전자산물이 생성된다. 본 연구에서는 pASPT2 cⅡ SD sequence 윗쪽에 있는 대부분의 5'untranslated sequence 가 제거된 pPLc-PT2는 pASPT2와 같은 수준의 사람 프리트롬빈 2를 생산하였다. 반면에 trp과 lac 프로모터로부터 유래된 tac 프로모터를 이용한 재조합 플라스미드들은 낮은 양의 프리트롬빈 2를 생산하는 것이 관찰되었다. SD sequence와 번역개시코돈 ATG 사이가 각각 6, 8, 10, 12는 뉴클리오티드가 떨어져 있고 뉴클리오티드 서열이 다른 tac 프로모타 융합플라스미드들을 제조하였다. SD sequence와 번역개시코돈 ATG 사이의 길이와 뉴크리오티드 서열의 변화는 각기 다른 플라스미드들에 의한 사람 프리트롬빈 2 발현 수준에 있어서 몇배의 차이를 야기시켰다. SD sequence가 번역개시코돈 ATG로부터 8 뉴클리오티드가 떨어진 ptacN_(8)PT2가 이들 플라스미드들 중에서 가장 높은 발현 수준을 나타내었는데, 그러나 pASPT2의 발현수준과 비교해 보면 몇 배가 낮은 수준이었다. 이 실험결과는 번역 조절이 각기 다른 플라스미드들이 의해 유전자 발현수준이 다르게 나타나는데 중요한 역할을 하고 있음을 보여주고 있다. In the previous report we have described the construction of the expression plasmid pASPT2 which produced high-level of human prethrombin 2 under the control of the thermoinducible bacteriophage λP_(L) promoter in E. coli, amounting to about 10% of the total bacterial proteins in the cells (Choi et al., 1989). _(p)PLc-PT2 was constructed in which the DNA region from -415 to -54 from the translation initiation codon ATG in _(p)A-SPT2 was substituted by seven nucleotides, AATTGGC. The recombinant plasmid produced the high-level of human prethrombin 2 comparable to _(p)ASPT2. In contrast to the high-level expression by _(p)ASPT2 and _(p)PLc-PT2 plasmids, we have observed reduced amounts of production by the recombinant plasmid utilizing the tac promoter derived from trp and lac promoters. A eries of tac promoter fusion plasmids were constructed in which the Shine-Dalgarno (SD) sequence were separated from the initiation codon ATG by 6, 8, 10, and 12 nucleotides with different nucleotide sequences. Variations of the distance and the nucleotide sequence between the SD sequence and the ATG codon leads to differences in the expression levels of human prethrombin 2 by the different plasmids. (ptac)_N_(8)-PT2 in which the SD sequence was separated from the ATG codon by 8 nucleotides was the highest producer among them, but produced less amount of the product than _(p)ASPT2 by at least several folds. Our results suggest that translational control plays an important role in the variations in the expression levels by the different plasmids.