鼠脱氧核糖核酸酶(DNase Ⅱα)基因的克隆及原核表达  

Cloning and prokaryotic expression of mouse DNase Ⅱα gene

作　　者：王卫芳[1,2,3] 刘明远[1] 欧阳红生[2] 吴秀萍[1] 王学林[1] 刘玛峰[1] 杨勇[1] 孙磊[1] 王子见[1] 张瑞[1] 于艳玲[1] 

机构地区：[1]吉林大学人兽共患病研究所人兽共患病教育部重点实验室,吉林长春130062 [2]吉林大学畜牧兽医学院,吉林长春130062 [3]长春中医药大学,吉林长春130017

出　　处：《中国兽医学报》2009年第6期752-755,共4页Chinese Journal of Veterinary Science

摘　　要：根据鼠脱氧核糖核酸酶Ⅱ(DNaseⅡ)基因设计引物,通过RT-PCR扩增出鼠DNaseⅡα基因片段,克隆到pMD-18T载体,然后转化至大肠杆菌JM109。经鉴定及序列测定,克隆基因与鼠DNaseⅡα基因序列完全一致,大小为1 008 bp。将重组质粒pMD-DNaseⅡα进行酶切后连接到原核表达载体pET-28a中,重组质粒经PCR和双酶切鉴定及序列分析正确后转化到大肠杆菌BL21(DE3)中,IPTG诱导表达,表达产物经SDS-PAGE检测,然后进行初步纯化,Western-blot鉴定。结果表明,本试验成功构建了pET-28a-DNaseⅡα表达载体;表达产物以包涵体形式存在,相对分子质量约为38 800;初步纯化的目的蛋白在SDS-PAGE图谱上呈单一条带,Western-blot鉴定能与抗体发生特异免疫反应。A pair of oligonucleotide primers were designed and syntheized according to the gene encoding mouse spleen DNase Ⅱα gene. After RT-PCR from total RNA of mouse spleen,the DNase Ⅱα gene was amplified by PCR and the purified PCR products were cloned into pMD-18T vector. After identification with restriction enzyme diges tion and sequence analysis, the positive plasmids pMD-DNase Ⅱα was cloned into prokaryotic expression vector pET- 28a and the recombinant plasmid pET-DNase Ⅱα was then transformed into the host E. coli BL21 （DE3）. The recombinant bacteria was induced by 1 mmol/L IPTG at 37℃ and the expression product was identified by SDS-PAGE and Western blot. Results showed that the expression product was the form of inclusion body about 388 000 and re acted with anti DNase Ⅱα antibody. The research laid a foundation of further study on function of DNase Ⅱα

关 键 词：脱氧核糖核酸酶Ⅱ(DNase Ⅱ) 克隆 原核表达 

分 类 号：Q78[生物学—分子生物学]