Isolation of Human Defensin Genes

人类防御素基因的分离

• 批准号: 6622321
• 负责人: KIRSTEN R JAGLO
• 金额: $ 4.64万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6622321
• 关键词: Saccharomyces cerevisiae; antibacterial agents; antifungal agents; biotechnology; biotherapeutic agent; cell growth regulation; cell line; chemical structure function; complementary DNA; defensins; fungal genetics; fungal proteins; fusion gene; gene mutation; gram negative bacteria; gram positive bacteria; human tissue; immunologic substance development /preparation; microorganism immunology; molecular cloning; nucleic acid sequence; polymerase chain reaction; postdoctoral investigator; trachea

DESCRIPTION (provided by applicant): Humans are remarkably good at fighting off attacks by pathogenic microorganisms unless they are immunosuppressed. Defensins are innate nonspecific, antimicrobial peptides that disable microbes and some viruses within 30 minutes. To date, six alpha- and three beta-defensins have been isolated in humans. Isolation of additional defensin genes and examination of their antimicrobial activity would increase understanding of the immune system and could be used in cancer treatment. Therefore, I will (1) develop screening conditions to isolate new defensins using the bakers yeast, Saccharomyces cerevisiae; (2) identify and characterize new defensin genes; and (3) further characterize known defensin genes. I will use a novel screening approach where peptides will be secreted from a high secretion strain of S. cerevisiae onto lawns of Gram-negative and Gram-positive bacteria. cDNAs from human tracheal cells will be cloned into a high copy vector containing both the promoter and secretion sequences from the yeast alpha-factor gene, MF-alpha-1. Yeast colonies that are surrounded by reduced bacterial growth will be isolated and the cDNA sequenced. Novel peptides will be examined for specific antibacterial and antifungal activity and tissue-specific expression in humans or mice. To better understand the mechanism of defensin action, known antimicrobial peptides will be randomly mutagenized by PCR, expressed and secreted by yeast (as above), then used to challenge Gram-negative and Gram-positive bacteria. Using optimal and sub-optimal conditions (e.g., high salt concentrations) peptides with increased antimicrobial activity will be isolated and sequenced. Through these experiments I hope to both isolate new defensins and increase understanding of known defensins.

描述（由申请人提供）：人类非常擅长战斗 除非受到免疫抑制，否则致病性微生物的攻击。 防御素是固有的非特异性抗菌肽，可使微生物禁用 还有一些病毒在30分钟内。迄今为止，六个α-和三个 β-防御素已在人类中孤立。隔离额外防御素 基因和检查其抗菌活性的检查将增加 了解免疫系统，可用于癌症治疗。 因此，我将（1）开发筛选条件以隔离新防御素 使用面包师酵母，酿酒酵母； （2）识别和表征 新的防御基因； （3）进一步表征已知的防御素基因。我会 使用一种新颖的筛选方法，其中将从高的肽中分泌 酿酒酵母的分泌菌株对革兰氏阴性和革兰氏阳性的草坪 细菌。来自人气管细胞的cDNA将被克隆到高拷贝中 载体包含酵母的启动子和分泌序列 α-因子基因，MF-Alpha-1。被减少包围的酵母菌菌落 细菌生长将被分离，并测序cDNA。新型肽会 检查特定的抗菌和抗真菌活性以及 人类或小鼠的组织特异性表达。更好地了解 防御素作用的机理，已知的抗菌肽将是随机的 由PCR诱变，用酵母表达和分泌（如上），然后曾经用过 挑战革兰氏阴和革兰氏阳性细菌。使用最佳和 次优的条件（例如，高盐浓度）肽增加 抗菌活性将被分离和测序。通过这些 我希望两个实验能够隔离新防御素并增加对 已知的防御素。