辐射诱导人TRAIL和内皮抑素双基因共表达载体的构建及鉴定  

Construction and identification of double-gene co-expression vector with radiation-inducible human TRAIL and endostatin

作　　者：李艳博[1,2] 郭彩霞[1,2] 龚平生[3] 刘扬[2] 梁硕[2] 王宏芳[2] 王剑锋[2] 龚守良[2] 

机构地区：[1]首都医科大学公共卫生与家庭医学学院,北京100069 [2]吉林大学公共卫生学院卫生部放射生物学重点实验室,吉林长春130021 [3]吉林大学分子酶学工程教育部重点实验室,吉林长春130023

出　　处：《吉林大学学报（医学版）》2010年第6期1007-1011,共5页Journal of Jilin University：Medicine Edition

基　　金：国家自然科学基金资助课题(30570546;30870747;30970681)

摘　　要：目的:构建辐射敏感Egr-1启动子诱导分泌型人肿瘤坏死因子相关凋亡诱导配体(TRAIL)和内皮抑素(endostatin)双基因共表达的载体pshuttle-Egr1-shTRAIL-shES。方法:利用聚合酶链式反应(PCR)方法从pMD19 T-endostatin载体上扩增得到分泌型endostatin基因,并连接到pMD19T载体上进行测序,然后利用基因重组技术构建Egr-1启动子转录调控分泌型TRAIL和endostatin双基因表达的重组质粒pshuttle-Egr1-shTRAIL-shES。结果:PCR扩增出的650bp片段经测序证实其序列与预期一致,说明获得的分泌型人endostatin基因正确;对pshuttle-Egr1-shTRAIL-shES及其构建过程中涉及到的多种中间载体进行PCR和酶切鉴定,结果均与预期完全一致,证实含有辐射敏感Egr-1启动子的分泌型TRAIL和endostatin双基因共表达重组质粒pshuttle-Egr1-shTRAIL-shES构建正确。结论:成功构建了辐射诱导表达的双基因共表达载体pshuttle-Egr1-shTRAIL-shES,为探讨TRAIL和endostatin双基因-放射治疗的抗肿瘤作用及其机制奠定了实验基础。Objective To construct a recombinant plasmid pshuttle-Egr1-shTRAIL-shES containing tumor necrosis factor-related apoptosis-inducing ligand （TRAIL）and endostatin double genes.Methods The secretary endostatin gene（shES）fragment was amplified from the pMD19T-endostatin vector by PCR.The shES gene was ligated to pMD19Tand sequenced.Finally,using the gene recombinant technique,the recombinant plasmid pshuttle-Egr1- shTRAIL-shES with radiation-inducible Egr1 promoter,secretary TRAIL and endostatin double-gene was constructed.Results The sequence of the shES gene was in concordance with that anticipated indicating shES gene was acquired successfully.Moreover,the results acquired by PCR and restrictive digestion identification of the recombinant plasmid pshuttle-Egr1-shTRAIL-shES and all the vectors refered to its construction confirmed that pshuttle-Egr1-shTRAIL-shES was constructed correctly.Conclusion The radiation-inducible double-gene co-expression vector pshuttle-Egr1-shTRAIL-shES is constructed successfully,which would set the experimental foundation for further study on the anti-tumor effect of TRAIL and endostatin double-gene-radiotherapy and its related mechanisms.

关 键 词：人肿瘤坏死因子相关凋亡诱导配体 内皮抑素 辐射 EGR-1启动子 

分 类 号：Q691[生物学—生物物理学] R730.55[医药卫生—肿瘤]