肿瘤睾丸抗原CT45-5基因小干扰RNA表达载体的构建及干扰效果鉴定  

Construction of Small Interfering RNA of Cancer Testis Antigen CT45-5 Gene and Assay of its Interfering Effect

作　　者：柯波[1,2] 林宇翔[1,2] 绳纪坡[1] 于芳[1] 罗维[1,2] 马祖兴[1,2] 胡宝成[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100850 [2]福建农林大学生命科学院,福建福州350002

出　　处：《生物技术通讯》2012年第2期180-182,共3页Letters in Biotechnology

基　　金：国家自然科学基金(31070760;30770651;30670616);国家重点基础研究发展计划(2007CB914604)

摘　　要：目的:构建肿瘤睾丸抗原CT45家族中5号成员基因(CT45-5)的小干扰RNA(siRNA),并检测其对HeLa及HeLa/Fhit细胞内源性CT45-5基因及外源脆性组氨酸三联体(Fhit)基因表达的干扰效果。方法:根据CT45-5基因序列,通过生物信息学方法对靶向CT45-5基因的siRNA进行预测,从中筛选出2对合理的CT45-5-siRNA,将其克隆到siRNA表达载体pSilencer 2.1-U6 Hygro中,转化大肠杆菌DH5α,挑取阳性克隆进行测序鉴定;将构建的CT45-5-siRNA重组载体转染人宫颈癌细胞HeLa及HeLa/Fhit细胞,Western印迹检测siRNA对HeLa及HeLa/Fhit细胞内源性CT45-5及外源Fhit基因表达的干扰效果。结果:构建了2个CT45-5-siRNA重组质粒,其中一条对CT45-5基因具有明显的干扰作用,并且Fhit基因表达也被抑制。结论:构建的CT45-5-siRNA为与CT45-5功能相关的基因研究奠定了基础。Objective： To construct small interfering RNA（siRNA） of cancer testis antigen CT45-5 gene and identify the interfering effect. Methods： The sequence of two CT45-5-siRNA were designed and inserted into the pSilencer 2.1-U6 Hygro vector. After confirmation of the constructs by DNA sequencing, human cervical cancer HeLa cells and HeLa/Fhit cells overexpressing fragile histidine triad （Fhit） gene were transfected with CT45-5-siRNA expression vectors. The interfering effect of CT45-5-siRNA was identified by Western blot. Results： Two CT45-5-siRNA expression vectors were obtained, and one of which could effectively inhibit the expression of CT45-5 and Fhit gene. Conclution： The CT45-5-siRNA expression vectors constructed can provide the foundation for further study on CT45-5 relative genes.

关 键 词：肿瘤睾丸抗原CT45家族5号成员基因 小干扰RNA 脆性组氨酸三联体 表达载体 

分 类 号：Q78[生物学—分子生物学]