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目的:鼻咽癌(nasopharyngeal carcinoma,NPC)高发于中国南方,是一种恶性度较高的肿瘤,与EB病毒关系非常密切。本研究利用前期已经设计和克隆好的EB病毒(EBV)已知和预计的编码基因作为cDNA探针,用于研制EBV微阵列。利用能高产EBV病毒颗粒的B95-8细胞作为检测对象,观测基因芯片检测的效果,已确定是否将来用于临床检测。方法:87个EBV基因探针通过一对设计于载体多克隆两端的引物进行PCR扩增。产物纯化后,探针被打印在Corning玻片上。通过与Cy3标记的B95-8细胞cDNA进行杂交反应,检测B95-8细胞内EBV基因表达。RT-PCR验证检测。结果:利用构建的EBV芯片,成功地在B95-8细胞中检测到了几乎所有的EBV潜伏基因的表达,随后RT-PCR验证了该结果。结论:我们成功地构建了EBV微阵列,且设计的EBV探针检测是有效的。但EBV微阵列是否可以用于鼻咽癌标本的检测还有待进一步证实。
OBJECTIVE: Nasopharyngeal carcinoma (NPC) is a highly malignant tumor with high incidence in southern China and has a close relationship with Epstein-Barr virus. In this study, a previously known and predicted EBV-encoded gene was designed and cloned as a cDNA probe for the development of EBV microarrays. The use of B95-8 cells capable of producing high EBV virions as the test object to observe the effect of the gene chip test has been determined whether it will be used for clinical testing in the future. Methods: Eighty-eight EBV gene probes were amplified by PCR using a pair of primers designed on both ends of the polyclonal vector. After the product has been purified, the probe is printed on a Corning glass slide. EBV gene expression in B95-8 cells was detected by hybridization with Cy3-labeled B95-8 cells cDNA. RT-PCR validation test. Results: Using the constructed EBV chip, we successfully detected the expression of almost all EBV latent genes in B95-8 cells. The results were confirmed by RT-PCR. Conclusion: We successfully constructed the EBV microarray and the designed EBV probe assay was effective. Whether EBV microarray can be used for the detection of nasopharyngeal carcinoma remains to be confirmed.