Purification, Characterization, and Molecular Cloning of A Viral Protease Inhibitor

病毒编码的蛋白水解酶对前体蛋白的裂解是许多动植物病毒合成功能性病毒蛋白的一种常见的蛋白质合成策略。阻止这些裂解过程可能会为控制这些病毒的复制提供一种特殊的手段。这项拟议的项目旨在研究南方大学生物学家在豌豆胚胎中发现的一种蛋白酶抑制剂。在体外蛋白质合成系统中，该抑制剂能完全阻止菜豆花叶病毒(CPMV)前体蛋白的蛋白降解。该计划是开发一种完全纯化该抑制剂的纯化程序，对该抑制剂进行生化表征，将该抑制剂的活性与合成肽的活性进行比较，确定该抑制剂在抗CPMV和敏感的豌豆植株中是否存在。并用DNA克隆和测序的方法测定该抑制物的氨基酸序列。这项研究的长期目标是全面了解该抑制剂的结构和功能特性，并将其作为研究利用蛋白酶抑制剂作为预防或治疗病毒疾病的抗病毒药物的可能性的模型。除了研究目标外，这一拟议项目的另一个目标是向南方大学和其他少数民族机构的少数族裔学生和教职员工提供接受研究培训的机会，并获得现代分子生物学技术，包括基因工程技术的经验。这一目标将通过研讨会和讲习班来实现。

Proteolytic cleavage of precursor proteins by virus coded proteases is a common protein synthesis strategy utilized by many animal and plant viruses for synthesis of functional viral proteins. Prevention of these cleavage processes may offer a specific means for controlling replication of these viruses. The proposed project is designed to investigate a protease inhibitor which Southern University biologists found to exist in cowpea embryos. This inhibitor can completely prevent proteolytic processing of the precursor proteins of cowpea mosaic virus (CPMV) in an in vitro protein synthesizing system. The plan is to develop a purification procedure for complete purification of the inhibitor, to characterize the inhibitor biochemically, to compare the activity of the inhibitor to the activities of synthetic peptides, to determine the existence or absence of this inhibitor in CPMV resistance and sensitive cowpea plants, and to determine the amino acid sequence of the inhibitor using DNA cloning and sequencing methods. The long range goals of this research are to gain a complete understanding of the structural and functional properties of this inhibitor, and to use this inhibitor as a model for examining the possibility of utilizing protease inhibitors as antiviral agents for prevention or treatment of viral diseases. Besides the research goals, an additional objective of this proposed project is to present an opportunity to minority students and faculty of Southern University and other minority institutions to receive research training and to gain experience in modern molecular biology techniques, including genetic engineering techniques. This objective will be accomplished through seminars and workshops.