含翻译增强序列的人canstatin杜氏盐藻叶绿体表达载体的构建  

Construction of chloroplast expression vector of Dunaliella salina for recombinant human canstatin with translation enhancer sequence

作　　者：李一帆[1] 崔柳青[2] 韩康[1] 薛乐勋[1] 

机构地区：[1]郑州大学生物工程系细胞生物学研究室,郑州450001 [2]河南工业大学生物工程学院,郑州450001

出　　处：《郑州大学学报（医学版）》2013年第5期581-583,共3页Journal of Zhengzhou University(Medical Sciences)

基　　金：国家自然科学基金资助项目31000580;河南工业大学基金资助项目2011BS017

摘　　要：目的:构建含翻译增强序列的人canstatin杜氏盐藻叶绿体重组表达载体。方法:采用RT-PCR的方法,获得含有人canstatin的cDNA片段以及分别在5'UTR下游和3'UTR上游添加了翻译增强序列(UUAACUUUA)的人canstatin cDNA片段,将得到的目的片段分别连接到PMD18-T载体上,利用该载体将目的片段连接到荧光素酶基因Lux Ct表达盒中并进行酶切鉴定。结果:RT-PCR分别得到约700 bp的cDNA片段并克隆到PMD18-T载体上。用PaeR7Ⅰ、SphⅠ双酶切Lux Ct质粒,得到2 100 bp和10 000 bp的2条片段。然后,将大片段回收后分别与上一步得到的cDNA片段进行连接,酶切鉴定结果显示含有翻译增强序列的人canstatin片段成功插入到Lux Ct质粒中。结论:成功构建了含有翻译增强序列的人canstatin杜氏盐藻叶绿体重组表达载体。Aim： To construct a chloroplast expression vector of Dunaliella salina for recombinant human canstatin with translation enhancer sequence .Methods：The cDNA fragments encoding the human canstatin , the human canstatin with translation enhancer sequence （ UUAACUUUA） in downstream of 5＇UTR and that with translation enhancer sequence （UUAACUUUA） in upstream of 3＇UTR were obtained by RT-PCR, respectively.Subsequently, the fragments were cloned into the PMD18-T vector and were identified by digestion of restriction enzymes , respectively .The correct fragments were subcloned into the expression cassette in Lux Ct ,and then the recombinant plasmid was identified by digestion of restriction enzymes.Results：The RT-PCR products of about 700 bp were cloned into the PMD18-T vector.Lux Ct plasmid digested by PaeR7Ⅰand SphⅠproduced two fragments , one was of 2 100 bp and the other was of 10 000 bp, and they were recy-cled and ligated with the cDNA fragments of canstatin .The results of enzyme digestion showed that canstatin fragments with translation enhancer sequence were correctly inserted into the Lux Ct plamid .Conclusion：The chloroplast expression vec-tor of Dunaliella salina for recombinant human canstatin with translation enhancer sequence has been constructed success -fully.

关 键 词：CANSTATIN 翻译增强序列 叶绿体 杜氏盐藻 

分 类 号：Q781[生物学—分子生物学]