hTERT启动子调控下CD137L表达载体的构建及其在卵巢癌细胞中的表达

目的研究hTERT启动子调控下CD137L表达载体的构建及其在人卵巢癌细胞系中的表达，探讨CD137L靶向基因治疗的可行性。方法采用RT-PCR法从K562细胞中克隆CD137L全长基因, 将其克隆到真核表达载体pcDNA3中，构建的质粒为pcDNA3-hCD137L，用Hind Ⅲ和Xba Ⅰ双酶切后将CD137L再定向插入pBTdel-279中，构建成hTERT启动子调控下CD137L表达载体pBTdel-279-hCD137L，采用酶切法和测序法鉴定。用Fugene6转染试剂盒将两种重组质粒分别瞬时转染人卵巢癌细胞株OVCR3和人胚肺成纤维细胞株HELF，RT-PCR和流式细胞仪检测CD137L的表达。结果测序证实所克隆的CD137L基因序列与GENEBANK中的序列相符。经限制性内切酶酶切，电泳后显示片段插入正确，证实构建成功。pcDNA3-hCD137L转染后OVCR3细胞和HELF细胞均可检测到CD137L的表达，但pBTdel-279-hCD137L转染后仅在OVCR3细胞中检测到其表达，在HELF细胞中无表达。流式细胞显示pBTdel-279-hCD137L转染效率低于pcDNA3-hCD137L。结论hTERT启动子调控下CD137L表达载体构建正确，并可在卵巢癌细胞中较高表达，为探讨靶向基因治疗提供了实验依据，但hTERT启动子活性仍低于CMV启动子

Construction and expression of the vector of CD137L under the control of the hTERT promoter in human ovarian tumor cell lines

ObjectiveTo construct an expression vector of CD137L under the control of hTERT promoter and to investigate the expression of this gene in human ovarian tumor cell lines and the possibility of the targeted gene therapy of CD137L. MethodsThe CD137L gene was cloned from the K562 cell lines and was inserted into the pcDNA3 vector to form a new plasmid named pcDNA3-hCD137L. The plasmid was digested by Hind Ⅲ and Xba Ⅰ, then the CD137L fragment was inserted into the pBTdel 279 plasmid to get a new plasmid pBTdel-279-hCD137L, in which the expression of CD137L was under the control of hTERT promoter. The new plasmid was identified by digestion of restriction enzyme and sequencing. pcDNA3-hCD137L and pBTdel-279-hCD137L plasmids were transfected into human ovarian tumor cell line OVCR3 and human embryonic lung fibroblast cell line HELF. The expression of hCD137L was determined by RT-PCR and flow cytometry. ResultsThe plasmids were properly constructed. Both OVCR3 and HELF cell lines can express hCD137L after transfection with pcDNA3-hCD137L plasmids, but only OVCR3 cell line can expression hCD137L gene after transfection with pBTdel-279-hCD137L plasmids. The results of flow cytometry showed that the transfection efficiency of pBTdel-279-hCD137L was much lower than that of the pcDNA3-hCD137L plasmid. ConclusionsThe expression vector of CD137L under the control of hTERT promoter is successfully constructed and the expression of hCD137L gene can be targeted in tumor cells by the hTERT promoter. But the activity of hTERT promoter is much lower than that of the CMV promoter. How to increase the activities of the hTERT promoter needs further investigation.