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-

本研究的目的是寻找新的致病基因，并分析其编码蛋白的生物学功能。检测氯化血红素、生长和氧气对基因表达的影响。然后，构建了文库，并对新的已知基因进行了分类。利用生物素标记的编码16S-23S rRNA基因的聚合酶链式反应产物和链霉亲和素磁珠，从总RNA中减去16S和23S rRNA。用大小选择的cDNA柱去除5S rRNA和tRNA。用检测基因和生物素标记的对照基因消减后，将得到的cDNA液插入到载体中，转化大肠杆菌。对插入的片段进行DNA序列分析和同源性搜索。并通过RT-PCR或Northern-blotting对同源基因进行了鉴定。本实验室已克隆的40k-Da外膜相关蛋白基因在早对数和晚对数阶段的消减文库中被检测到。因此，我们通过插入诱变的方法构建了牙龈假单胞菌突变株，并对其进行了鉴定。该突变株与革兰氏阳性菌的聚集性、自身聚集性、与人上皮细胞的粘附性以及血凝、溶血素、RGP和KGP等蛋白酶活性均显著降低。SDS-PAGE和Western-blotting分析结果表明，突变株菌毛未在细胞表面表达。因此，突变体中这些致病力的降低可能是通过改变外膜蛋白和菌毛等细胞表面成分来实现的。综上所述，该消减技术在原核生物致病力检测中具有广阔的应用前景。

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.