分泌型碱性磷酸酶报告基因重组载体pSEAP—IQCE3’UTR的构建  

Construction of Recombinant Plasmid pSEAP-IQCE3＇UTR

作　　者：王三虎[1] 马云[1] 秦凌雪[1] 何彬彬[1] 李斌元[1] 苏泽红[1] 何淑雅[1] 

机构地区：[1]南华大学生物化学与分子生物学教研室,湖南省衡阳市421001

出　　处：《医学分子生物学杂志》2012年第5期355-359,共5页Journal of Medical Molecular Biology

基　　金：湖南省自然科学基金（No.12JJ6073）

摘　　要：目的构建分泌型碱性磷酸酶（secretedalkalinephosphatasereportergene。SEAP）报告基因重组载体pSEAP—IQCE3’UTR，为鉴定FXR1P是否是通过与IQCE的3’UTR发生相互作用，进一步探索FXR1P的功能奠定基础。方法首先设计目的基因片段IQCE3’UTR的PCR引物，然后通过RT—PCR扩增加CE基因3’UTR基因片段，酶切扩增的片段和报告基因空载体，并将空载体去磷酸化，去磷酸化后的空载体与目的基因片段进行连接反应，将连接产物转化人大肠埃希菌JM109，最后筛选阳性克隆，PCR鉴定插入序列的正反。结果构建了分泌型碱性磷酸酶报告基因重组载体pSEAP—IQCE3’UTR，经酶切鉴定、正反鉴定和测序分析可知重组载体构建成功。结论成功构建了分泌型碱性磷酸酶报告基因重组载体pSEAP—IQCE3’UTR，为进一步探索FXR1P的功能及其在脆性X综合征中的发病机理有着重要的意义。Objective To construct the recombinant vector pSEAP-IQCE3＇UTR containing se- creted alkaline phosphatase reporter gene and explore whether FXR1P plays its role by interacting with IQCE3＇UTR. Methods The PCR primers for the target gene fragment of IQCE3 ＇UTR were de- signed, and then the IQCE3＇UTR gene fragments were amplified by RT-PCR. The restriction endo- nuclease Xba I was used for digestion of the amplified fragments and the reporter gene empty vec- tor. The empty vectors were dephosphorylated and then connected with the target fragment. The resul- ting product was transformed into E. coli JM109, and the positive clones were selected. The se- quence was detected by PCR. Results Restriction endonuclease digestion, positive and negative i- dentification and sequencing analysis showed that the recombinant vector pSEAP-IQCE3＇UTR contai- ning secreted alkaline phosphatase reporter gene was successfully constructed. Conclusion The successfully constructed recombinant vector pSEAP-IQCE3＇UTR is of great significance in further ex- ploring the function of FXR1P and its pathogenesis in fragile X syndrome.

关 键 词：FXR1P IQCE SEAP 脆性X综合征 

分 类 号：Q75[生物学—分子生物学] Q78