VEGF-C基因3′端未翻译区对荧光素酶表达的影响  

Influence of 3' untranslated region of vascular endothelial growth factor-C gene on the expression of luciferase

作　　者：王俊[1] 郭燕[1] 章必成[2] 关华军[1] 陈正堂[1] 

机构地区：[1]第三军医大学新桥医院全军肿瘤研究所,重庆400037 [2]广州军区武汉总医院肿瘤科

出　　处：《解放军医学杂志》2007年第9期922-925,共4页Medical Journal of Chinese People's Liberation Army

基　　金：国家自然科学基金资助项目(30371586)

摘　　要：目的构建小鼠血管内皮生长因子-C(VEGF-C)基因3′端未翻译区(3′UTR)的荧光素酶表达载体,利用双荧光报告系统观察VEGF-C基因3′UTR对荧光素酶基因表达的影响。方法通过PCR分别扩增小鼠Lewis肺癌细胞VEGF-CcDNA全长3′UTR(429bp)和一段312bp的编码区序列(CR),采用基因工程方法将PCR产物克隆至pTA2克隆载体,随后再亚克隆至荧光素酶报告基因载体(pGL3-Promoter)荧光素酶编码基因下游的XbaⅠ酶切位点,使用LipofectamineTM2000真核转染小鼠Lewis肺癌细胞,化学发光法检测荧光素酶的活性,定量RT-PCR检测荧光素酶mRNA水平。结果PCR成功扩增出与预期长度相符的小鼠VEGF-CCR(312bp)和VEGF-C3′UTR(429bp)。经酶切、测序鉴定,小鼠VEGF-C基因3′UTR和CR均插入到pGL3-Promoter的XbaⅠ位点,插入序列碱基组成和插入方向均正确,获得载体pGL3-VEGF-C3′UTR和pGL3-VEGF-CCR。荧光素酶报告系统和定量RT-PCR检测显示,与pGL3-Promoter组比较,pGL3-VEGF-C3′UTR转染组荧光素酶活性和mRNA水平均显著降低,而pGL3-VEGF-CCR组与pGL3-Promoter组之间无显著性差异。结论VEGF-C基因3′UTR可以抑制荧光素酶的表达。Objective To construct a luciferase gene expression vector containing full-length 3＇ untranslated region （3＇ UTR） of mouse vascular endothelial growth factor-C （VEGF-C） gene, and to observe the effects of VEGF-C 3＇UTR on luciferase gene expression by a double-fluorescence report system. Methods Polymerase chain reaction （PCR） was used to amplify VEGF-C 3＇UTR and a 312hp VEGF-C coding region （CR） fragment from full-length VEGF-C cDNA in mouse Lewis lung cancer cells. The luciferase expression vectors containing VEGF-C 3＇UTR or VEGF-C CR were constructed by subcloning the PCR products to luciferase reporter vector pGL.3-Prornoter using gene engineering technology, and then they were transfected to mouse Lewis lung carcinoma cells by LipofectamineTM 2000. The activities and rntLNA expression of luciferase were detected by Dual-Luciferase Reporter System and quantitative RT-PCR, respectively. Results Mouse VEGF-C 3＇UTR （429bp） and VEGF-C CR （312bp） were successfully amplified by PCR. The VEGF-C 3＇UTR and VEGF-C CR fragments were successfully inserted into pTA2 vector, and then subcloned to pGL3-Promoter vector at XbaI site by using restriction endonucleases analysis. The DNA sequences and insertion orientation of PCR products were all correct by sequencing analysis. The resulted luciferase expression plasmids were named pGL3-VEGF-C 3＇UTR and pGL3-VEGF-C CR, respectively. Dual-I,uciferase Reporter System detection and quantitative RT-PCR showed that in Lewis lung carcinoma cells, the activities of luciferase and expression of luciferase mRNA in the pGL3-Promoter group were higher than that in the pGL3,-VEGF-C 3＇UTR group, and there was no significant difference between pGL3- VEGF-C CR group and pGL3-Promoter group. Conclusion VEGF-C 3＇UTR can inhibit luciferase gene expression.

关 键 词：血管内皮生长因子C 3'未翻译区 荧光素酶 淋巴管生成 

分 类 号：R734.2[医药卫生—肿瘤]