宫颈癌细胞中辅酶Ⅱ依赖性视黄醇脱氢-还原酶及其选择性剪接亚型.docVIP

宫颈癌细胞中辅酶Ⅱ依赖性视黄醇脱氢/还原酶及其选择性剪接亚型 　【摘要】 目的：构建宫颈癌细胞中辅酶Ⅱ依赖性视黄醇脱氢/还原酶(NRDR)及选择性剪接亚型NRDRB1真核表达载体，并转染HeLa细胞表达融合蛋白和native蛋白。方法：分别以pDONR_NRDR、pDONR_NRDRB1载体为模板，用PCR方法扩增两端带有酶切位点的NRDR及NRDRB1编码区序列，酶切后连接到pCDNA3、pCMV_Myc真核表达载体上，经酶切、测序鉴定后，转染HeLa细胞16?h，Western blot检测蛋白表达情况，蛋白质谱分析鉴定表达蛋白。结果：成功构建NRDR、NRDRB1真核表达载体，转染HeLa细胞后，可表达带有Myc标签的蛋白和不带标签的native蛋白；质谱分析结果进一步证实所表达的蛋白为NRDR和NRDRB1。结论：获得了在真核细胞中表达的NRDR、NRDRB1蛋白，为进一步研究其功能提供了实验材料。 【关键词】 辅酶Ⅱ依赖性视黄醇脱氢/还原酶 选择性剪接 宫颈癌 真核表达 ［Abstract］Objective： To construct the expression plasmids of NADP(H)_dependent retinol dehydrogenase/reductase(NRDR)and NRDRB1， and to express the proteins in mammalian cells. Methods： The coding regions of NRDR and NRDRB1 were amplified from pDONR vectors. The fragments of NRDR and NRDRB1 were ligated with mammalian expression vector pCDNA3 and pCMV_Myc after restriction enzyme. HeLa cells were transfected with the recombinant expression plasmids， and the target proteins were detected by Western blot. Results： The recombinant plasmids of pCDNA3_NRDR， pCDNA3_NRDRB1， pCMV_Myc_NRDR and pCMV_Myc_NRDRB1 were obtained and identified by restriction enzyme and sequencing. After transfection 16 hours， the target proteins were detected by Western blot. The expressed proteins were identified by the mass spectrometry. Conclusion： The recombinant NRDR and NRDRB1 from human cervical carcinoma cells can be expressed in mammlian cells. ［Key Words］NADP(H)_dependent retinol dehydrogenase/reductase；alternative splicing；cervical carcinoma；recombinant expression 辅酶Ⅱ依赖性视黄醇脱氢/还原酶(NRDR)具有较强的视黄醇氧化和视黄醛还原活性，存在于哺乳动物多种组织中，是参与全反式视黄酸或称维甲酸(atRA)代谢通用途径的关键酶。参与atRA代谢的酶可通过影响atRA与其前体或类似物在细胞内的转化而影响其生物学效应，因而与肿瘤发生等生物学过程密切相关。研究表明，视黄醇及其它视黄酸类物质对于维持正常宫颈上皮功能、抑制宫颈癌组织的生长具有重要作用［1～3］，视黄醇缺乏或视黄酸物质代谢紊乱是除人乳头瘤病毒感染外引发宫颈癌的另一重要病因［4， 5］，我们从人宫颈癌细胞中克隆鉴定了一种视黄醇脱氢/还原酶_辅酶Ⅱ依赖性视黄醇脱氢/还原酶(NADP_dependent retinol dehydrogenase/reductase，NRDR)，同时发现了一种新的NRDR选择性剪接亚型NRDRB1［6］(Genbank accession number DQ325464)。为深入研究宫颈癌细胞中NRDR、NRDRB1的活性和功能，本研究构建了哺乳动物细胞表达载体，对上述两种蛋白进行表达。 1材料与方法 11细胞株及主要试剂 HeLa细