摘要
目的 :探讨大肠癌相关基因 ST13上游 5 '近端调控区 (- 5 95～ + 74 )中增强子碱基序列。方法 :设计系列缺失 PCR引物 ,扩增 ST13基因 5 '近端碱基序列片段 ,p GEMT- EASY克隆、测序 ,再亚克隆到 p GL 2系列瞬间报告载体上 ,等量转染 SW6 2 0细胞 ,检测荧光酶活性。结果 :系列扩增碱基序列片段 6 6 9bp、2 6 3bp、16 3bp对p GL2 - Basic中的荧光素酶基因均具有较强的启动表达作用 ,片段 10 1bp、4 7bp则几乎没有启动下游基因表达的作用。而 10 1bp片段与系列报告载体 p GL 2重组的分子 ,在其中含有启动子的报告基因表达载体中具有明显的增强作用 (P<0 .0 1) ,但作用强度小于阳性对照 p GL2 - Control(P<0 .0 5 )。结论 :位于 ST13基因上游 5 '近端调控区的碱基序列 10 1bp片段 (- 5 95～ - 494 ) 。
Objective: To study the base sequence of an enhancer in up stream 5' flank near regulation region (from -595 to +74) of human colorectal cancer related gene ST13. Methods: Several deletion PCR primers were designed. Amplified DNA fragments of ST13 gene 5' flank near region were cloned with pGEMT EASY vector and sequenced; then subcloned into several pGL2 report vectors respectively. Equal quantitative recombined DNA was transfected into SW620 cell lines and the luciferase activity was checked. Results: Several amplified base sequence fragments (669 bp,263 bp,163 bp) in pGL2 Basic all enhanced and promoted luciferase gene expression strongly. The 47 bp and 101 bp fragments didn't promote luciferase gene expression. 101 bp fragment recombined with pGL2 Promoter enhanced luciferase gene expression distinctly ( P <0.01), but the effect was less strong than the positive pGL2 Control( P <0.05). Conclusion: The base sequence 101 bp (from -595 to -494) in up stream 5' flank near regulation region of colorectal cancer related gene ST13 is an enhancer regulating gene transcript.
出处
《浙江大学学报（医学版）》
CAS
CSCD
2004年第5期385-389,共5页
Journal of Zhejiang University(Medical Sciences)
基金
国家自然科学基金 ( 30 1710 34)
