中文摘要：

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Ni^2＋-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。

英文摘要：

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.