사람의 침샘에서 발견된 새로운 항균단백질의 분자적 분리 및 동정 Molecular Cloning of a Novel Antimicrobial Protein from Human Submandibular Gland
pp.275-286
In the previous molecular cloning study from human salivary gland cDNA l ibrary a novel clone (C77-091) was known as a candidate gene for antimicrobial protein by GenBank database search and RNA in situ hybridization. This study is aimed to identify the molecular characteristics of C77-091 protein, which showed an antimicrobial activity on E.coli, thereby named as salivary antimicrobial protein (SAMP). SAMP consisted of a typical hydrophobic amino acid rich domain in the N-terminus, a cluster of basic amino acids, carbohydrate attachment site, a possible transglutaminase catalyzed cross-linking site, and multiple consensus sequences of phosphorylation site in the C-terminus. Western blot analysis of human organs and tissue with the monospecific antibody to the synthetic SAMP peptide showed strong interacting protein from the extracts from submandibular gland and parotid saliva but absent in the mixed saliva, and the immunohistochemical staining detected a strong positive regions in the secretory granules in the luminal cytoplasm of interlobular ductal cells of salivary gland. The SAMP was also distributed in the human sebaceous gland and prostate. These data suggest that C77-091 named SAMP gene is a novel antimicrobial protein in human salivary gland, which may play a role for the innate immunity by protecting and stabilizing the mucosal epithelium to maintain homeostasis of oral mucosa.
II. 재료 및 방법
1. Reverse transcription polymerase chainreaction (RT-PCR) 및 intron의 존재 확인
2. 단백질 motif 검색 및 가상 단백질 구조
3. 항체 제작 및 정제
4. 면역조직화학적 염색
5. Western blot
6. 재조합단백질 생산
7. 항균작용 실험 (Antimicrobial assay
III. 연구 결과
1. 염기서열과 아미노산 서열 및 단백질 특성
2. 가상 단백질 구조
3. 중합효소사슬반응 (PCR)
4. 면역조직화학적 염색
5. Western blot
6. 재조합단백질 생산
7. 항균 작용 실험 (Antimicrobial assay)
IV. 총괄 및 고찰
V. 참고문헌