A construction of cDNA library and categorization of genes by subtraction in prokaryote - A gene cloning of new known gene associated with pathogenic factors in P. gingivalis-

原核生物cDNA文库构建及基因消减分类-牙龈卟啉单胞菌致病因子相关新已知基因的基因克隆-

基本信息

批准号：
09470405
负责人：
ABIKO Yoshimitsu
金额：
$ 3.78万
依托单位：
Nihon University School of Dentistry at Matsudo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

项目摘要

A purpose of this study were a search of new pathogenic genes and a analysis of the biological function of the coded protein in Porpyromonas gingivalis. The effects of hemin, growth or oxygen to gene expression were examined. Then, the cDNA library was constructed and the new known genes were categorized. For purification of mRNA, the 16S and 23S rRNA were subtracted from a total RNA by using the biotin-labeled PCR product which is coded 16S-23S rRNA gene and streptoavidin magnet beads. The 5S rRNA and tRNA were removed by using a cDNA column with size-selection. After subtraction with tester cDNA and biotin-labeled control cDNA, the resulting cDNA solution was inserted into the plasmid and transformated into E. coli. The inserted fragments were analyzed the DNA sequence and searched the homology. Moreover, the homologous genes were identified by RT-PCR or Northern-blotting. A 40k-Da outer membrane-associated protein gene, which was cloned previously in our laboratory, was detected in subtracted cDNA library between early-log phase and late-log phase. Hence, we constructed the P. gingivalis mutant by insertion mutagenesis and characterized. The mutant has led to diminished remarkably aggregation with Gram-positive bacteria, the autoaggregation, adherence to human epithelial cells, and activities of hemagglutination, hemolysin, protease like RGP and KGP. The results in SDS-PAGE and Western-blotting analysis were suggested the fimbriae of the mutant did not expressed on the cell surface. So, the reduction of these pathogenicity in the mutant might be occurred by change of cell surface components like outer membrane proteins and fimbriae and so on. In conclusion, this subtraction technology should have a wide range of application to the detection of the pathogenicity in prokaryote.

本研究的目的是寻找新的致病基因并分析牙龈卟啉单胞菌编码蛋白的生物学功能。研究了血红素、生长或氧气对基因表达的影响。然后，构建cDNA文库并对新的已知基因进行分类。为了纯化mRNA，通过使用编码16S-23S rRNA基因的生物素标记的PCR产物和链霉亲和素磁珠从总RNA中减去16S和23S rRNA。使用具有大小选择功能的 cDNA 柱去除 5S rRNA 和 tRNA。用测试cDNA和生物素标记的对照cDNA进行扣除后，将所得的cDNA溶液插入质粒中并转化至大肠杆菌中。对插入的片段进行DNA序列分析并搜索同源性。此外，通过RT-PCR或Northern印迹鉴定同源基因。在对数早期和对数晚期的消减cDNA文库中检测到了我们实验室先前克隆的40k-Da外膜相关蛋白基因。因此，我们通过插入诱变构建了牙龈卟啉单胞菌突变体并进行了表征。该突变体导致与革兰氏阳性菌的聚集、自聚集、对人上皮细胞的粘附以及血凝、溶血素、蛋白酶（如RGP和KGP）的活性显着减弱。 SDS-PAGE和Western-blotting分析结果表明突变体菌毛在细胞表面不表达。因此，突变体中这些致病性的降低可能是通过改变细胞表面成分（如外膜蛋白和菌毛等）而发生的。总之，该消减技术在原核生物致病性检测中应该具有广泛的应用前景。