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

摘要

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.%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DHSα,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CT4 5-5-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.