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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
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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.
出处 《中国生物制品学杂志》 CAS CSCD 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)
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 Ornithine decarboxylase antizyme-1 (OAZ1) Functional gene SOE PCR Prokaryotic expression
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