摘要
目的:运用差异显示技术初步筛选肾虚型支气管哮喘的相关基因,为探讨肾虚证的相关基因表达谱打下基础。方法:用限制性酶切片段差异显示技术(RFDD-PCR),收集支气管哮喘肾虚证患者治疗前后外周血,分离纯化其总RNA、合成双链cDNA、TaqI限切酶酶切、在酶切位点两端连上接头、用特异配对于接头的引物进行降落PCR扩增、变性聚丙烯酰胺凝胶电泳、银染、回收差异条带并重扩增、反向斑点杂交、测序、生物信息学分析。结果:共找到三十六条差异条带,其中二十五条能用斑点杂交验证,选取共性差异条带三条进行测序,结果与基因库比较未发现同源序列。结论:三条基因片段可能是与支气管哮喘肾虚证相关的新基因。
Objective: Screening relative genes of patients with asthma of kidney-deficiency by DD-PCR technique to lay the foundation for relative gene expression profile of kidney deficiency syndrome. Methods: With RFDD-PCR, we colleated the patients' peri-blood before and after treatment, isolated and purified the blood for its NA respectively, treated with Dnasel and transcripted to cDNA. Then we cut it with Taqi and connected the adaptors, and next amplified PCR with primers which annealed to the adaptors specially. The experiment still continued in the following steps: electrophoresing on 7 percent polyacry lamide gel, staining with AgNo_3, comparing the display profile to find the differential bands and to cut them, reamplifying the differeatial bands, dotting the products of PCR on the nylon membrane, hybridizing with the cDNAs which were reversely transcripted from total RNA, sequencing the positive bands and blasting them with the data in Genebank. Results: We found 36 differential bands from the gel and 25 of them could be tested by hybridization. Three of the differencial expressions are the same among three patients. Sequencing these three and blasting with Genbank, we haven't found homologous consequence with it. Conclusion: The three gene's fragments may be new related genes of asthma due to kidney deficiency.
出处
《河南中医学院学报》
2005年第3期13-15,共3页
Journal of Henan University of Chinese Medicine
关键词
肾虚型支气管哮喘
外用血mRNA差异
基因表达谱
asthma with kidney deficiency
mRNA differential display
genes' expression profiles
