| ObjectiveThe purpose of the current study was to identify the differential genes in Porphyromonas gingivalishighly toxic strain W83 and minimally toxic strain ATCC 33277. The differential subtraction library was established. The positive clones were identified by PCR and then sequenced, and searched homologically. Comparative whole - genome analysis of highly toxic and minimally toxic strains of Porphyromonas gingivalis has identified the clustering of genes that were present in W83 but divergent in or absent from ATCC 33277. Then, we screened virulence genes of wild strain Porphyromonas gingivalis by genechip. These genes may provide an important clue for studying the mechanism of occurrence and development of periodontal disease.Methods1. Study population:There were 50 subjects who were included in the case group and 20 subjects who were included in the healthy group in the study. The subjects age - and gender - matched with healthy periodontal conditions were enrolled. The subjects were excluded if they smoked and received systemic professional periodontal therapy or taken any medicine during 3 months prior to the study. They all knew the fact and consented.2. Measurement of clinical periodontal index;The study selected the lesion and non - lesion status in chronic periodontitis and the healthy status in healthy people. Clinical parameters were measured andrecorded by a single examiner, which included bleeding on probing (BOP) , clinical attachment loss ( CAL) , probing depth ( PD ) , and tooth movement (TM).3. Sample collection and bacterial culture:Subgingival plaque samples were taken by sterile subgingival curette. Every subject was placed into bacterium refer fluid (TD) , and then cultured.4. Detection P. gingivalis : Genomic DNA was extracted with phenol and chloroform from plaque and thallus. Subsequently, extracted DNA was amplified with 16S rRNA species - specific primer, and was detected on 1.5% agarose gel electrophoresis.5. Suppression subtractive hybridization;Using suppression subtractive hybridization ( SSH ) to compare Porphyromonas gingivalis highly toxic strain W83 (tester) and minimally toxic strain ATCC 33277 ( driver). The chromosomal DNAs were purified from Porphyromonas gingivalis W83 and Porphyromonas gingivalis ATCC 33277, and digested by restriction enzyme Rsa I . The tester DNA samples were separated and ligated with adaptor 1 and adaptor 2R. Two subtractive hybridization and PCR profile were performed. Tester - specific DNAs also were selectively amplified.6. Carrier conjugation and transformation:The mixture of subtracted DNA fragments were ligated with pMD - 18T vector and transformed to competent cells E. coli JM109. The different subtraction library was established. The positive clones were identified by PCR and then se-quenced, and searched homologically.7. Opposite experiment;Using suppression subtractive hybridization ( SSH ) to compare Porphyromonas gingivalis minimally toxic strain ATCC 33277 (tester) and highly toxic strain W83 (driver).8. Dot blot analysis: ,Digested DNA of Porphyromonas gingivalis minimally toxic strain ATCC 33277 and highly toxic strain W83 were labed by Digoxin. Through dot blot a-nalysis confirmed that all these fragments are present in Porphyromonasgingivalis W83(ATCC 33277) but absent from ATCC 33277( W83)9. Detection by DNA microarray:The wild strains Porphyromonas gingivalis were used as probes. The genes acquired by Suppression subtractive hybridization were probed. Detecting DNA of the wild strains Porphyromonas gingivalis by DNA microarray Hybridization .10. The repetition of genechips test;20 random samples were tested by dot blot analysis and repeat tests of five times were done for each genechip and the results were any lysis.11. Statistical analysis;The study use SPSS12.0 statistical software. Chi square test and Spearman Correlation analysis were employed to this study. Significance level: P < 0. 05 is considered statistically significant, P < 0. 01 is considered higher statistically significant.Results1. Suppression subtractive hybridization results :The Rsa I - digested DNA were ligated with adaptor 1 and adaptor 2R. We performed the ligation efficiency to verify that at least 50% of tester DNA fragments have adaptors on both ends. Through two subtractive hybridization and two PCR amplification ,we obtained the products. The experimental PCR subtraction products with some distinct bands were different from unsubtraction products. The distinct bands may were enriched in Porphyromonas gingivalis highly toxic strain W83 which were absent from Porphyromonas gingivalis ATCC 33277. We performed the subtraction efficiency anylysis. The subtraction products were ligated with pMD - 18T vector and transformed to competent cells E. coli JM109. The differential subtraction library was established. 194 positive clones were obtained by blue - white selection and were identified by PCR and then 165 clones have products 100 ~500bp,110 clones were sequenced and searched homologically.2. BLAST results;Subtractive library which had high subtractive efficiency was successfullyset up and 36 positive clones were screened by SSH. The fragments from 88bp to 372bp were enriched in Porphyromoruis gingivalis highly toxic strain W83 sequences which were absent from P. gingivalis ATCC 33277. Through dot blot a-nalysis confirmed that all these fragments are present in Porphyromonas gingivalis W83 but absent from ATCC 33277. The CenBank homology search indicated that among them, Pg0836>Pg0838^Pg0839 and Pgl436 are associated with two paralogous regions of the chromosome;two framgments PG0043 and PG0108 are associated with evasion of P. gingivalis W83, PG0540 was releted to antibiotic resistance and the products of PG0724, PG1055, PG1701 are virulence and acquisition of peptides. Some genes were associated with evasion of Porphyromonas gingivalis W83;Another gene was related to antibiotic resistance and the products of some genes were virulence and acquisition of peptides.3. Dot blot analysis results:Through dot blot analysis confirmed that all these 36 fragments are present in P. gingivalis W83 but absent from ATCC 33277.4. Opposite experiment results:Subtractive library which has high subtractive efficiency was successfully set up and 140 positive clones were obtained by blue - white selection and were identified by PCR and then 119 clones that had products 100 ~700bp were se-quenced and searched homologically. 62 positive clones were screened by SSH. The fragments from 125 - 632 bp were enriched in Porphyromonas gingivalis minimally toxic strain ATCC 33277 sequences which were absent from Porphyromonas gingivalis. W83. Through dot blot analysis confirmed that all these fragments were present in Porphyromonas gingivalis ATCC 33277 but absent from W83.5. Detection by DNA microarray:( 1 ) The hybridization results of disease region and normal region P. gingivalisThrough Chi - square analysis, highly statistically significant differences were observed about eleven fragments results . ( seven results P < 0.05 ,four results P < 0. 01 ) Other results observed were no statistic differences.(2) The relations of hybridization results with clinical indexCorrelation analysis showed that the rate of positive about fragments related to PDNCAL and TM( P < 0.05 )6. Veracity and repetition of Hybridization20 random samples were tested by dot blot analysis and repeat tests five times were done for each genechip. The hybridization results of microarray have good Veracity and repetition.Conclusion |
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