论文部分内容阅读
目的构建人PDLIM4基因启动子荧光素酶报告基因重组质粒pGL3-promoter,检测其在不同肿瘤细胞中的表达。方法据Genebank中人PDLIM4基因启动子序列分别设计上下游引物,扩增其启动子片段,并插入到pGL3-Basic报告基因载体,分别得到含有3kb和1.2kb的PDLIM4基因启动子的两个报告基因质粒pGL3-PD-LIM4-3kb和pGL3-PDLIM4-1.2kb。序列为1.2kb的启动子利用Erase-a-base试剂盒,经外切酶Ⅲ从5’端逐步酶切得到5个含不同长度启动子序列的报告基因载体:pGL3-PDLIM4-1.2D1、pGL3-PDLIM4-1.2D2、pGL3-PDLIM4-1.2D3、pGL3-PDLIM4-1.2D4、pGL3-PDLIM4-1.2D5。将构建的报告基因载体分别瞬时转染以下细胞株:Hela、MDA-MB231、KB、PC-3、LNCaP,检测其荧光素酶表达活性。结果所构建质粒经酶切、测序鉴定,其片段长度及序列均正确。转染结果显示,长片段的3kb和1.2kb启动子序列在所测试的细胞株中均有表达,但在人激素非依赖前列腺癌PC-3细胞株、人激素依赖前列腺癌LINCaP细胞株中表达较低。而不同长度的启动子报告基因质粒转染PC-3、LNCaP的结果显示,pGL3-PDLIM4-1.2kb表达活性最强,pGL3-PDLIM4-970bp表达活性最弱。结论成功构建了7个分别含有不同长度的PDLIM4启动子报告基因载体,其在不同的肿瘤细胞株中均有不同程度的表达,而在前列腺癌细胞中表达较低。
Objective To construct pGL3-promoter, a luciferase reporter gene of human PDLIM4 promoter, to detect its expression in different tumor cells. Methods The upstream and downstream primers of PDLIM4 promoter were designed according to the sequence of human PDLIM4 promoter in Genebank. The promoter fragment was amplified and inserted into the pGL3-Basic reporter vector to obtain two reporter genes containing PDLIM4 promoter of 3kb and 1.2kb, respectively Plasmids pGL3-PD-LIM4-3kb and pGL3-PDLIM4-1.2kb. The sequence of 1.2kb promoter using Erase-a-base kit, exonuclease Ⅲ from the 5 ’end by step digestion of five different length promoter sequence containing the reporter vector: pGL3-PDLIM4-1.2D1, pGL3 -PDLIM4-1.2D2, pGL3-PDLIM4-1.2D3, pGL3-PDLIM4-1.2D4, pGL3-PDLIM4-1.2D5. The constructed reporter gene vectors were transiently transfected into the following cell lines: Hela, MDA-MB231, KB, PC-3 and LNCaP, respectively, and their luciferase expression activities were tested. Results The constructed plasmids were identified by restriction enzyme digestion and sequencing. The length and sequence of the fragments were correct. The transfection results showed that the 3kb and 1.2kb promoter sequences of the long fragments were all expressed in the cell lines tested, but were expressed in human hormone-independent prostate cancer PC-3 cell line and human hormone-dependent prostate cancer cell line LINCaP Lower. The results of PCNA-3 and LNCaP transfected with reporter gene plasmids of different lengths showed that the expression of pGL3-PDLIM4-1.2kb was the strongest and the expression of pGL3-PDLIM4-970bp was the weakest. Conclusion Seven PDLIM4 promoter vectors containing different lengths of PDLIM4 promoter were successfully constructed, which were expressed in different degrees in different tumor cell lines, but lower in prostate cancer cells.