【摘要】 背景与目的:在前期研究中发现,16HBE-C细胞表达变化的基因中有未知基因参与,因其与BPDE有关,所以命名为brg(anti-BPDErelatedgene)基因。本研究应用cDNA末端快速扩增法(rapidamplificationofcDNAends,RACE)扩增此未知基因的全长,以进行下一步的基因功能研究。材料与方法:应用cDNA末端快速扩增法(RACE技术),对3’和5’端分别设计了梯度PCR,巢式PCR等优化程序,以获得特异的产物,然后对特异产物直接测序,将测序结果进行生物信息学分析,基因拼接等获得新基因brg全长。结果:3’和5’端均成功获得特异性产物,3’RACE测序为394bp,有明显的PolyA加尾信号,有编码区的终止密码子;5’RACE测序为1017bp,有起始密码子ATG,其中197bp为3’和5’全长共有序列。将3’和5’序列拼接,结果全长1214bp,生物信息学分析brg基因阅读框1032bp,编码344个氨基酸。结论:所得结果与设计一致,说明所采用的技术路线是简便可行的,brg基因可能在反式二羟环氧苯并(a)芘的致癌机制中起重要作用。更多还原

【Abstract】 BACKGROUNG&AIM: Previous studies showed a gene fragment was identified as being over_expressed in16HBE_C cells when compared to16HBE cells,which is named brg(anti_BPDE related gene).The aim of this study was to obtain the full_length cDNA of brg based on its EST fragment and to provide basic data for the functional research of the novel gene. MATERIAL AND METHODS: Based on the rapid amplification of cDNA ends(RACE)method,gradient and nested PCR protocols were used to obtain full_length cDNA in order to get a discrete and bright band on an agarose gel electrophoresis.The nested PCR products were then sequenced and the bioinformatic analysis was carried out. RESULTS: Full_length cDNA of the gene was successfully cloned by both3’_end RACE and5’_end RACE.The cDNA had a total length of1214bp.Sequence analysis of the full_length cDNA of the gene revealed that the cDNA encoded an open reading frame of1032bp;with the deduced protein containing344amino acids. CONCLUSION: The general and simple protocol was useful for cloning of full_length gene based on EST fragment.The results suggest that brg gene may be involved in carcinogenesis during anti_BPDE related tumor development.更多还原