RFDD-PCR技术用于构建证的相关基因表达谱初探

目的　运用限制性酶切片段差异显示 (RFDD -PCR)技术初步筛选支气管哮喘肾虚证的相关基因 ,以冀探讨构建中医证的相关基因表达谱的一种研究方法。方法　应用RFDD -PCR技术 ,收集支气管哮喘肾虚证患者治疗前后外周血 ,分离其总RNA ,进行DNaseI处理、合成双链cDNA、TaqI限切酶酶切、在其两端连上接头、特异配对于接头的引物来降落PCR扩增、变性聚丙烯酰胺凝胶电泳、AgNO3 染色、回收差异条带并重新PCR扩增、反向斑点杂交、测序、生物信息学分析。结果　共找到 36条差异条带 ,其中 2 5条能用斑点杂交验证 ,选取 3条测序 ,结果与基因库比较未发现同源序列。结论　 3条基因片段可能是与支气管哮喘肾虚证相关的新基因 ;RFDD -PCR技术不失为构建中医证的相关基因表达谱的一种好方法。

Primary discuss of related genes'''' expressional chart of syndromes constructed with RFDD-PCR technique

Objective It is to discuss a method of constructing related gene expressional chart of syndrome via primary filtrating related gene of kidney asthenia in bronchial asthma with RFDD-PCR technique.Methods The peripheral blood of the patients with kidney asthenia in bronchial asthma was collected before and after treatment. Then with RFDD-PCR technique, its total RNA was separated and DNase Ⅰwas disposed, double chain cRNA was composed, TaqI enzyme was cut, connected and amplified with primers which was specially paired to the joint, descent PCR was amplified, denatured polyacrylamide gel electrophoresis was did and stained with AgNO_3, the different strip were recovered and PCR was amplified again, reverse fleck was hybridized, sequence was determined and the biological informatics was analyzed. Results 36 different strips were found. 25 of them could be validated with fleck hybridized. Three of them were chosen and determined the sequence and none homologous was found compared with the gene bank.Conclusion The three gene fragments may be new related genes of kidney asthenia in bronchial asthma. The RFDD-PCR technique is a good method of constructing related gene expressional chart of syndrome.