小鼠牙本质涎磷蛋白成熟蛋白编码区cDNA克隆与部分序列分析

Cloning and partially sequencing of mouse dentin sialophosphoprotein encoding mature protein

目的 克隆小鼠牙本质涎磷蛋白 (dentinsialophosphoprotein ,DSPP)成熟蛋白编码区基因。方法 用异硫氰酸胍一步法从昆明新生小鼠磨牙牙胚组织中抽提总RNA ,用Oligo(dt)作引物逆转录合成牙胚cDNA ,然后利用PCR方法 ,从cDNA中扩增出小鼠DSPP成熟区的基因片段 (约 3kbp) ,将所得基因片段插入pBluescriptKS质粒载体 ,转化到大肠杆菌XL1 Blue后挑选阳性克隆 ,提取重组质粒DNA ,通过限制性酶切和核苷酸序列分析鉴定阳性克隆。结果 重组质粒pBS DSPP的酶切图谱和部分序列分析结果与国外文献报道一致。结论 克隆到小鼠DSPP成熟编码区基因 ,正在进行该基因的表达和活性鉴定。

Objective Cloning and partially sequencing of mouse dentin sialophosphoprotein (DSPP) encoding mature protein Methods Total RNA was extracted from the tooth germs of newborn mouse by acid guanidinium thiocyanata phenol chloroform method, the desired DNA product was obtained from the total RNA by RT PCR with the primers including Oligo(dt) and two gene specific primers The segment (about 3 Kbp) was inserted into pBluescript vector and the interesting plasmid was transformed into E Coli host strain XL1 Blue The double stranded DNA of the positive clone was analyzed by restriction endonuclease mapping and DNA sequencing Results The restriction endonuclease map and sequence of mouse DSPP encoding mature protein were consistent with those of the published Conclusion The mouse DSPP cDNA encoding mature protein was obtained for further study