小鼠鸟氨酸脱羧酶抗酶1功能基因的克隆及原核表达  

Cloning and Prokaryotic Expression of Functional Gene of Mouse Ornithine Decarboxylase Antizyme-1

作　　者：刘梦瑶[1] 韩钰[1] 蔡富强[1] 何玲[1] 王艳林[1] 

机构地区：[1]三峡大学分子生物学研究所,湖北宜昌443002

出　　处：《中国生物制品学杂志》2010年第2期138-141,149,共5页Chinese Journal of Biologicals

基　　金：国家自然科学基金资助项目(30772590)

摘　　要：目的克隆小鼠鸟氨酸脱羧酶抗酶1(OAZ1)功能基因,原核表达并纯化OAZ1重组蛋白。方法采用RT-PCR法从小鼠黑色素瘤细胞总RNA中扩增OAZ1基因,通过重叠延伸PCR技术构建无需移码即可全长翻译的功能基因,并构建重组表达质粒pET15b/OAZ1-T,转化大肠杆菌BL21(DE3),经IPTG诱导表达。表达的重组蛋白经Ni2+-NTA亲和层析纯化后,进行SDS-PAGE和Western blot分析。结果重叠PCR法扩增出692bp的OAZ1功能基因,重组表达质粒经酶切及测序鉴定正确,重组蛋白可在大肠杆菌中以包涵体形式高效表达。纯化的重组蛋白纯度可达79.96%,且可与抗His标签抗体特异性结合。结论已成功克隆了小鼠OAZ1功能基因,原核表达并纯化了重组OAZ1蛋白。Objective To clone the functional gene of mouse ornithine decarboxylase antizyme-1（OAZ1）, express in prokaryotic cells and purify the recombinant OAZ1 protein. Methods OAZ1 gene was amplified from the total RNA of mouse melanoma B16-F1 cells by RT-PCR, and a functional gene of which the full-length could be translated without frameshifting was obtained by SOE PCR and used for construction of recombinant plasmid pET15b / OAZ1-T which was transformed to E. coli BL21（DE3）for expression under induction of IPTG. The expressed recombinant protein was purified by Ni^2＋-NTA affinity chromatography and identified by SDSPAGE and Western blot. Results The functional gene of OAZ1, at a length of 692 bp, was amplified by SOE PCR, identified as target gene by restriction analysis and sequencing and expressed in a form of inclusion body in E. coli. The purified recombinant protein reached a purity of 79. 96% and showed specific reaction with antibody against His label. Conclusion The functional gene of mouse OAZ1 was successfully cloned and expressed in prokaryotic cells, and recombinant OAZ1 protein was purified.

关 键 词：鸟氨酸脱羧酶抗酶1 功能基因 重叠延伸PCR 原核表达 

分 类 号：Q786[生物学—分子生物学]