Construction and application of cloning vector which carries signal sequence of Escherichia coli.

本项目的目的是阐明大肠杆菌外膜蛋白之一--抗洗涤剂磷脂酶A(DR-Pholipase A)的定位和加工的分子机制。我们还尝试构建了利用DR-磷脂酶A.1信号序列的克隆载体。DR-磷脂酶A前体分子的生化和遗传学分析：克隆了DR-磷脂酶A的结构基因pldA。对pldA的体外产物进行了分析，发现其相对分子质量略高于体内产物。在其他实验中，在编码DR-磷脂酶A信号序列的DNA区域引入了定点突变，导致一个氨基酸插入。突变后的pldA基因产酶量较少。这些结果有力地表明了DR-磷脂酶A前体分子的存在，突变的pldA基因产生的DR-磷脂酶A量较少，可能是由于加工步骤效率低下所致。克隆载体的构建：在编码DR-磷脂酶A信号序列的DNA区域后立即引入含有多克隆位点的合成DNA片段，将pldA基因的启动子分别替换为三个功能良好的启动子(tac、Consensus和lambda PL)。载体应用：将合成的溶菌酶福曼胃泌素基因克隆到新的克隆载体上。对携带重组质粒的大肠杆菌菌株进行了人类蛋白的表达检测。到目前为止，人们观察到了成熟形式的人溶菌酶的产生和积累，但产量很低。必须对表达式进行进一步的改进。

The aim of this project was to clarify a molecular mechanism of location and processing of one of E. coli outer membrance protein, detergent resistant phospholipase A (DR-phospholipase A). We also attempted to construct a cloning vector which utilized the signal sequence of DR- phospholipase A.1. Biochemical and genetical analysis of DR-phospholipase A precursor molecule:The structure gene of DR-phosphclipase A, pldA, was cloned. The in vitro product of pldA was analyzed and was found to bear a molecular weight slightly higher than that generated in vivo< In other experiment, a site-directed mutagenesis, which resulted in one amino acid insertgion, was introduced into the DNA region coding for the signal sequence of the DR-phospholipase A. The mutated pldA gene was found to produce less amount of enzyme. These results strongly suggested the existence of a precursor molecule of DR-phospholipase A. Less amount of production of DR-phospholipase A from mutated pldA gene might be due to the inefficient processing step.2. Construction of cloning vector:A synthetic DNA fragment containing multi-cloning sites was introduced immediately after the DNA region which coded for the signal sequence of DR- phospholipase A. The promoter of pldA gene was then replaced by three well-characterized promoters (tac, consensus, and lambda PL), respectively.3. Application of vectors:Synthetic genes coding for fuman urogastron of lysozyme were cloned onto these novel cloning vectors. E. coli strains bearing recombinant plasmids were examined for the expression of human proteins. So far, production and accumulation of a mature form of human lysozyme was observed, however, the yield was low. Further improvement in the expression must be performed.