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目的:构建牙龈卟啉单胞菌(简称P.gingivalis)全基因组规模的微阵列比较基因组杂交(简称arrayCGH)芯片平台,分析不同菌株间基因组的差异,为后续检测临床分离株的毒力基因,进一步阐述牙周炎发病机制提供依据。方法:综合已经全基因组测序的12株P.gingivalis菌的序列信息,设计探针序列,定制芯片。提取P.gingivalis高毒力株W83和低毒力株ATCC 33277的基因组DNA,利用array-CGH检测其全基因组的差异DNA片段,并运用PCR验证结果的准确性。结果:Array-CGH结果显示两组菌株间存在几个连续片段的拷贝数不同。P.gingivalis W83的部分特异片段参与编码毒力致病因子。在P.gingivalis ATCC 33277的特有片段中,部分编码蛋白可增强细菌表明粘附力,使其具有高粘附力。从中挑选12个基因进行PCR差异性验证,证实与芯片结果一致。结论:用array-CGH芯片的方法来分析全基因组拷贝数的变化具有分辨率高,能精确定位异常片段的特性。本文成功构建了array-CGH技术检测P.gingivalis不同毒力株基因差异的平台,为后续比较临床分离株的差异DNA片段,筛查毒力基因,深入阐述牙周炎的发病机制奠定基础。
OBJECTIVE: To construct genome-wide microarray array CGH chip platform of P. gingivalis (P. gingivalis) for the purpose of analyzing the genome differences of different strains and for the subsequent detection of virulence genes of clinical isolates, Further elaborate the pathogenesis of periodontitis provide basis. Methods: Based on the sequence information of 12 strains of P. gingivalis which had been sequenced in the whole genome, the probe sequences were designed and customized. Genomic DNA of P. gingivalis high virulent strain W83 and virulent strain ATCC 33277 was extracted and the whole genome DNA was detected by array-CGH. The accuracy of the results was verified by PCR. Results: The results of Array-CGH showed that the copy numbers of several consecutive fragments were different between the two strains. Partial fragments of P.gingivalis W83 are involved in the coding of virulence factors. In the unique fragment of P. gingivalis ATCC 33277, partially-encoded proteins enhance the adhesion of bacteria to bacteria, giving them high adhesion. 12 genes were selected for PCR differential validation, confirmed the same chip results. Conclusion: The array-CGH chip method to analyze the whole genome copy number changes with high resolution, can accurately locate the characteristics of abnormal fragments. In this paper, we successfully constructed the platform of array-CGH to detect the genetic differences of different virulence strains of P. gingivalis, and laid the foundation for the further comparison of DNA fragments of different clinical isolates, screening virulence genes and further elaborating the pathogenesis of periodontitis.