Screening For Trypsin—like Enzyme Gene By The Metagenomics And The Subgingival Plaque Bacterial Diversity Analysis Of Chronic Periodontitis

Periodontitis is a gum swelling, bleeding, pus overflow atrophy gingival meat, gingival leakage and loose teeth, which is characterized by a group of multifactorial disease, the main pathological changes contain gingival inflammation and bleeding、 periodontal pocket formation, alveolar boneloss, loosening of teeth and shift result in tooth loss, which is one of the periodontal disease. Periodontitis is one of the common diseases in the human mouth. There is currently80-97%of Chinese adults suffer from varying degrees of periodontal disease according to the third national oral health epidemiological survey. Chronic periodontitis is a kind of periodontitis that is a set of complex causes inflammation of the devastating disease. The clinical manifestations of bleeding gums, periodontal pocket formation, alceolar bone loss and tooth fall off. Epidemiological study estimated that over four million American over the age of45who suffer from this disease.The part1of this thesis:Microbial metagenomics technology was be used in moderate chronic periodontitis subgingival plaque. First, we extract the total DNA of moderate chronic subgingival plaque. The metagenomics library was constructed by the pWEB-TNCTM Fosmid Cloning Kit. The total clone number in the library is6500, and the average insert fragment is30K bp. The total insert fragment is about200M bp. The fosmid DNA were respectively extracted from12clones which are closen in random, and there were digested by BamH I. The diversity of metagenomics library was verified by the digestion pattern of12clones, BANA method was used to screen trypsin-like enzyme from the metagenomic. A clone can express trypsin-like enzyme by BANA experience, which is named E5. The16S rDNA sequence of positive clone E5was indentified by NICB datebase. The result show the DNA sequence of clone E5has97.5%of the similarity with pseudomonas, we get the trypsin-like enzyme gene by subcloning. Then the trypsin-like protease gene is identified by NCBI datebase, the result show that the gene has75%of the similarity with trypsin-like serine protease.The part2of this thesis:Amplifed Ribosomal DNA Restriction Analysis (ARDRA) and Single-Strand Conformation Polymorphism (SSCP) technololy are used to analysis the bacterial communities of patients. The Amplifed Ribosomal DNA Restriction Analysis(ARDRA) was applied to analysis the bacterial communities of patients with moderate chonic periodontitis subgingival plaque、saliva and gingival crevicular fluid. We are establishment of subgingival plaque, saliva and gingival crevicular fluid16S rDNA library. There are61kinds OUTs in subgingival plaque16S rDNA library which has92clones, there are34kinds OUTs in saliva16S rDNA library which has96clones, and there are42kinds OUTs in gingival crevicular fluid16S rDNA library which has94clones. The diversity index of the culture independent method is Shannon-wienner (H):3.92; simpson index (D):0.938; richness (R):38.23; which is higher than the other simples. All the results show that there is abundant functional, structural and genetical bacterical diversity in the chonic moderate periodontitis subgingival plaque, which provide the informational for the follow-up metagenomic research. Single-Strand Conformation Polymorphism (SSCP) was used to analysis the bacterial diversity in the subgingival plaque of healthy person and patients with moderate and severe chronic periodontitis. The whole DNA was extracted from subgingival plaque of patients, and V4-V5region of16S rDNA was amplified. We got the single-stranded of PCR product by digested and denatured. Then we select the predominant strains by analysising the relative brightness and abundance of the bands on the polyacrylamide gel. The result show that Prevotella intermedia、Porphyromonas gingivalis、Campylobacter rectus、Veillonella parvula、 Fusobacterium nucleatum those are the several major pathogen of the CP which have a higher detection rate. And the detection rate is also high as the occurrence and development of CP, which confirms the pathogenicity of these types of bacteria.